Isolated mammalian monocyte cell genes; related reagents

ABSTRACT

Nucleic acids encoding various monocyte cell proteins from a primate, reagents related thereto, including specific antibodies, and purified proteins are described. Methods of using said reagents and related diagnostic kits are also provided.

[0001] This filing is a conversion of, and claims benefit of priorityto, provisional U.S. Patent Applications U.S. S No. 60/032,252, filedDec. 6, 1996; U.S. S No. 60/033,181, filed Dec. 16, 1996; and U.S. S No.60/041,279, filed Mar. 21, 1997, each of which is incorporated herein byreference.

FIELD OF THE INVENTION

[0002] The present invention contemplates compositions related to genesfound in monocyte cells, cells which function in the immune system.These genes function in controlling development, differentiation, and/orphysiology of the mammalian immune system. In particular, theapplication provides nucleic acids, proteins, antibodies, and methods ofusing them.

BACKGROUND OF THE INVENTION

[0003] The circulating component of the mammalian circulatory systemcomprises various cell types, including red and white blood cells of theerythroid and myeloid cell lineages. See, e.g., Rapaport (1987)Introduction to Hematology (2d ed.) Lippincott, Philadelphia, Pa.; Jandl(1987) Blood: Textbook of Hematology, Little, Brown and Co., Boston,Mass.; and Paul (ed.) (1993) Fundamental Immunology (3d ed.) RavenPress, NY

[0004] Monocytes are phagocytic cells that belong to the mononuclearphagocyte system and reside in the circulation. See Roitt (ed)Encyclopedia of Immunology Academic Press, San Diego. These cellsoriginate in the bone marrow and remain only a short time in the marrowcompartment once they differentiate. They then enter the circulation andcan remain there for a relatively long period of time, e.g., a few days.The monocytes can enter the tissues and body cavities by the processdesignated diapedesis, where they differentiate into macrophages andpossibly into dendritic cells. In an inflammatory response, the numberof monocytes in the circulation may double, and many of the increasednumber of monocytes diapedese to the site of inflammation.

[0005] Antigen presentation refers to the cellular events in which aproteinaceous antigen is taken up, processed by antigen presenting cells(APC), and then recognized to initiate an immune response. The mostactive antigen presenting cells have been characterized as themacrophages, which are direct developmental products from monocytes;dendritic cells; and certain B cells.

[0006] Macrophages are found in most tissues and are highly active ininternalization of a wide variety of protein antigens andmicroorganisms. They have a highly developed endocytic activity, andsecrete many products important in the initiation of an immune response.For this reason, it is believed that many genes expressed by monocytesor induced by monocyte activation are likely to be important in antigenuptake, processing, presentation, or regulation of the resulting immuneresponse.

[0007] However, monocytes are poorly characterized, both in terms ofproteins they express, and many of their functions and mechanisms ofaction, including their activated states. In particular, the processesand mechanisms related to the initiation of an immune response,including antigen processing and presentation, remain unclear. Theabsence of knowledge about the structural, biological, and physiologicalproperties of these cells limits their understanding. Thus, medicalconditions where regulation, development, or physiology of antigenpresenting cells is unusual remain unmanageable.

SUMMARY OF THE INVENTION

[0008] The present invention is based, in part, upon the discovery ofvarious genes isolated from activated monocytes. These molecules havebeen designated FDF03 (a type I transmembrane protein with Ig-likeextracellular portion); YE01 (an Fc gamma/alpha-like receptor); andKTE03 class (cell surface receptors exhibiting Ig-like domains),represented by YYB01, YYB04 related, KLM63, KLM66, and KLM67embodiments.

[0009] The invention provides various compositions of matter selectedfrom: a substantially pure or recombinant FDF03 protein or peptideexhibiting at least about 85% sequence identity over a length of atleast about 12 amino acids to mature SEQ ID NO: 2 or 4; a naturalsequence FDF03 of SEQ ID NO: 2 or 4; a fusion protein comprising FDF03sequence; a substantially pure or recombinant YE01 protein or peptideexhibiting at least about 85% sequence identity over a length of atleast about 12 amino acids to mature SEQ ID NO: 6, 8, or 10; a naturalsequence YE01 of SEQ ID NO: 6, 8, or 10; a fusion protein comprisingYE01 sequence; a substantially pure or recombinant KTE03 protein orpeptide exhibiting at least about 85% sequence identity over a length ofat least about 12 amino acids to SEQ ID NO: 12, 14, 16, 18, 20, or 22; anatural sequence KTE03 of SEQ ID NO: 12, 14, 16, 18, 20, or 22; or afusion protein comprising KTE03 sequence. Preferably, the substantiallypure or isolated protein comprises a segment exhibiting sequenceidentity to a corresponding portion of a FDF03, YE01, or KTE03, wherein:the homology is at least about 90% identity and the portion is at leastabout 9 amino acids; the homology is at least about 80% identity and theportion is at least about 17 amino acids; or the homology is at leastabout 70% identity and the portion is at least about 25 amino acids. Inother forms, the invention provides such composition of matter, whereinthe: FDF03 comprises a mature sequence of Table 1; YE01 comprises amature sequence of Table 2; KTE03 comprises a mature sequence of Table3; or the protein or peptide: is from a warm blooded animal selectedfrom a mammal, including a primate or rodent; comprises at least onepolypeptide segment of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or22; exhibits a plurality of portions exhibiting the identity; is anatural allelic variant of FDF03, YE01, or KTE03; has a length at leastabout 30 amino acids; exhibits at least two non-overlapping epitopeswhich are specific for a mammalian FDF03, YE01, or KTE03; exhibits asequence identity at least about 90% over a length of at least about 20amino acids to a rodent FDF03, YE01, or KTE03; exhibits at least twonon-overlapping epitopes which are specific for a primate FDF03, YE01,or KTE03; exhibits a sequence identity at least about 90% over a lengthof at least about 20 amino acids to a primate FDF03, YE01, or KTE03; isglycosylated; has a molecular weight of at least 7 kD with naturalglycosylation; is a synthetic polypeptide; is attached to a solidsubstrate; is conjugated to another chemical moiety; is a 5-fold or lesssubstitution from natural sequence; or is a deletion or insertionvariant from a natural sequence.

[0010] Other compositions include those comprising: a sterile FDF03protein or peptide; the FDF03 protein or peptide and a carrier, whereinthe carrier is: an aqueous compound, including water, saline, and/orbuffer; and/or formulated for oral, rectal, nasal, topical, orparenteral administration; a sterile YE01 protein or peptide; the YE01protein or peptide and a carrier, wherein the carrier is: an aqueouscompound, including water, saline, and/or buffer; and/or formulated fororal, rectal, nasal, topical, or parenteral administration; a sterileKTE03 protein or peptide; or the KTE03 protein or peptide and a carrier,wherein the carrier is: an aqueous compound, including water, saline,and/or buffer; and/or formulated for oral, rectal, nasal, topical, orparenteral administration.

[0011] In fusion protein embodiments, the invention provides those whichcomprise: mature protein sequence of Table 1, 2, or 3; a detection orpurification tag, including a FLAG, His6, or Ig sequence; or sequence ofanother cell surface protein.

[0012] Various kits include those comprising a protein or polypeptide,and: a compartment comprising the protein or polypeptide; and/orinstructions for use or disposal of reagents in the kit.

[0013] Antibodies and binding compounds include those comprising anantigen binding portion from an antibody, which specifically binds to anatural FDF03, YE01, or KTE03 protein, wherein: the protein is a primateprotein; the binding compound is an Fv, Fab, or Fab2 fragment; thebinding compound is conjugated to another chemical moiety; or theantibody: is raised against a peptide sequence of a mature polypeptideof Table 1, 2, or 3; is raised against a mature FDF03, YE01, or KTE03;is raised to a purified FDF03, YE01, or KTE03; is immunoselected; is apolyclonal antibody; binds to a denatured FDF03, YE01, or KTE03;exhibits a Kd to antigen of at least 30 μM; is attached to a solidsubstrate, including a bead or plastic membrane; is in a sterilecomposition; or is detectably labeled, including a radioactive orfluorescent label. A kit comprising the binding compound is providedincluding, e.g., the binding compound and: a compartment comprising thebinding compound; and/or instructions for use or disposal of reagents inthe kit. Preferably, the kit is capable of making a qualitative orquantitative analysis.

[0014] Various other compositions include those comprising: a sterilebinding compound; or the binding compound and a carrier, wherein thecarrier is: an aqueous compound, including water, saline, and/or buffer;and/or formulated for oral, rectal, nasal, topical, or parenteraladministration.

[0015] Nucleic acid embodiments include an isolated or recombinantnucleic acid encoding a protein or peptide or fusion protein asdescribed, wherein: the protein is from a mammal, including a primate;or the nucleic acid: encodes an antigenic peptide sequence of Table 1,2, or 3; encodes a plurality of antigenic peptide sequences of Table 1,2, or 3; exhibits at least about 80% identity to a natural cDNA encodingthe segment; is an expression vector; further comprises an origin ofreplication; is from a natural source; comprises a detectable label;comprises synthetic nucleotide sequence; is less than 6 kb, preferablyless than 3 kb; is from a mammal, including a primate; comprises anatural full length coding sequence; is a hybridization probe for a geneencoding the protein; or is a PCR primer, PCR product, or mutagenesisprimer.

[0016] Various cells are provided, including those comprising adescribed recombinant nucleic acid. Preferably, the cell is: aprokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; aninsect cell; a mammalian cell; a mouse cell; a primate cell; or a humancell. Kits with such nucleic acids include those with the nucleic acidand: a compartment comprising the nucleic acid; a compartment furthercomprising a FDF03, YE01, or KTE03 protein or polypeptide; and/orinstructions for use or disposal of reagents in the kit. Preferably, thekit is capable of making a qualitative or quantitative analysis.

[0017] Other nucleic acids include those which: hybridize under washconditions of 300° C. and less than 2M salt to the coding portions ofSEQ ID NO: 1 or 3; hybridize under wash conditions of 30° C. and lessthan 2 M salt to the coding portions of SEQ ID NO: 5, 7, or 9; hybridizeunder wash conditions of 300° C. and less than 2M salt to the codingportions of SEQ ID NO: 11, 13, 15, 17, 19, or 21; exhibit at least about85% identity over a stretch of at least about 30 nucleotides to aprimate FDF03; exhibit at least about 85% identity over a stretch of atleast about 30 nucleotides to a primate YE01; or exhibit at least about85% identity over a stretch of at least about 30 nucleotides to aprimate KTE03. In preferred embodiments, the wash conditions are at 450°C. and/or 500 mm salt; or at 550° C. and/or 150 mM salt; or the identityis at least 90% and/or the stretch is at least 55 nucleotides; or theidentity is at least 95% and/or the stretch is at least 75 nucleotides.

[0018] The invention further provides a method of modulating physiologyor development of a cell or tissue culture cell comprising contactingthe cell with an agonist or antagonist of a FDF03, YE01, or KTE03. Inpreferred embodiments, the cell is a leukocyte, and the antagonist is toYE01 and is a monoclonal antibody which binds to DLAIR-1.

DETAILED DESCRIPTION

[0019] Outline

[0020] I. General

[0021] II. Definitions

[0022] III. Nucleic Acids

[0023] IV. Making Proteins

[0024] V. Antibodies

[0025] VI. Purified Proteins

[0026] VII. Physical Variants

[0027] VIII. Binding Agent:Monocyte Protein Complexes

[0028] IX. Uses

[0029] X. Kits

[0030] XI. Binding Partner Isolation

[0031] I. General

[0032] The present invention provides DNA sequences encoding mammalianproteins expressed on monocytes. For a review of monocytes and theirfunctions, see, e.g., Gallin, et al. (eds. 1988) Inflammation: BasicPrinciples and Clinical Correlates Raven Press, NY; van Furth (ed. 1985)Mononuclear Phagocytes: Characteristics. Physiology and FunctionMartinus Nijhoff, Dordrecht, Netherlands.

[0033] Specific human embodiments of these proteins are provided below.The descriptions below are directed, for exemplary purposes, to humanmonocyte genes, but are likewise applicable to structurally, e.g.,sequence, related embodiments from other sources or mammalian species,including polymorphic or individual variants. These will include, e.g.,proteins which exhibit a relatively few changes in sequence, e.g., lessthan about 5%, and number, e.g., less than 20 residue substitutions,typically less than 15, preferably less than 10, and more preferablyless than 5 substitutions. These will also include versions which aretruncated from full length, as described, and fusion proteins containingsubstantial segments of these sequences.

[0034] II. Definitions

[0035] The term “binding composition” refers to molecules that bind withspecificity to a these monocyte proteins, e.g., in an antibody-antigeninteraction, or compounds, e.g., proteins, which specifically associatewith the respective protein. Typically, the association will be in anatural physiologically relevant protein-protein interaction, eithercovalent or non-covalent, and may include members of a multiproteincomplex, including carrier compounds or dimerization partners. Themolecule may be a polymer, or chemical reagent. A functional analog maybe a protein with structural modifications, or may be a wholly unrelatedmolecule, e.g., which has a molecular shape which interacts with theappropriate interacting determinants. The variants may serve as agonistsor antagonists of the protein, see, e.g., Goodman, et al. (eds.) (1990)Goodman & Gilman's: The Pharmacological Bases of Therapeutics (8th ed.)Pergamon Press, Tarrytown, N.Y.

[0036] The term “binding agent:monocyte protein complex”, as usedherein, refers to a complex of a binding agent and the monocyte protein.Specific binding of the binding agent means that the binding agent has aspecific binding site that recognizes a site on the respective monocyteprotein. For example, antibodies raised to the monocyte protein andrecognizing an epitope on the monocyte protein are capable of forming abinding agent:monocyte protein complex by specific binding. Typically,the formation of a binding agent:monocyte protein complex allows themeasurement of monocyte protein in a mixture of other proteins andbiologics. The term “antibody:monocyte protein complex” refers to abinding agent:monocyte protein complex in which the binding agent is anantibody. The antibody may be monoclonal, polyclonal or even an antigenbinding fragment of an antibody.

[0037] “Homologous” nucleic acid sequences when compared, exhibitsignificant similarity. The standards for homology in nucleic acids areeither measures for homology generally used in the art by sequencecomparison and/or phylogenetic relationship, or based upon hybridizationconditions. Hybridization conditions are described in greater detailbelow.

[0038] An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA,or a mixed polymer, which is substantially separated from othercomponents which naturally accompany a native sequence, e.g., proteinsand flanking genomic sequences from the originating species. The termembraces a nucleic acid sequence which has been removed from itsnaturally occurring environment, and includes recombinant or cloned DNAisolates and chemically synthesized analogs or analogs biologicallysynthesized by heterologous systems. A substantially pure moleculeincludes isolated forms of the molecule. An isolated nucleic acid willgenerally be a homogeneous composition of molecules, but will, in someembodiments, contain minor heterogeneity. This heterogeneity istypically found at the polymer ends or portions not critical to adesired biological function or activity.

[0039] As used herein, the term “monocyte protein” shall encompass, whenused in a protein context, a protein having amino acid sequences asshown in SEQ ID NO: 2 or 4; 6, 8, or 10; or 12, 14, 16, 18, 20, or 22,or a significant fragment of such a protein. It refers to a polypeptidewhich interacts with the respective monocyte protein specific bindingcomponents. These binding components, e.g., antibodies, typically bindto the monocyte protein with high affinity, e.g., at least about 100 nM,usually better than about 30 nM, preferably better than about 10 nM, andmore preferably at better than about 3 nM.

[0040] The term “polypeptide” or “protein” as used herein includes asignificant fragment or segment of said monocyte protein, andencompasses a stretch of amino acid residues of at least about 8 aminoacids, generally at least 10 amino acids, more generally at least 12amino acids, often at least 14 amino acids, more often at least 16 aminoacids, typically at least 18 amino acids, more typically at least 20amino acids, usually at least 22 amino acids, more usually at least 24amino acids, preferably at least 26 amino acids, more preferably atleast 28 amino acids, and, in particularly preferred embodiments, atleast about 30 or more amino acids. Fragment or size limitationsapplicable for comparison to one group, e.g., to the FDF03, do notnecessarily imply similar size limitations on fragments for the others.

[0041] A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, for example, products made by transforming cells with anynon-naturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site. Alternatively, it is performed tojoin together nucleic acid segments of desired functions to generate asingle genetic entity comprising a desired combination of functions notfound in the commonly available natural forms. Restriction enzymerecognition sites are often the target of such artificial manipulations,but other site specific targets, e.g., promoters, DNA replication sites,regulation sequences, control sequences, or other useful features may beincorporated by design. A similar concept is intended for a recombinant,e.g., fusion, polypeptide. Specifically included are synthetic nucleicacids which, by genetic code redundancy, encode polypeptides similar tofragments of these antigens, and fusions of sequences from variousdifferent species variants.

[0042] “Solubility” is reflected by sedimentation measured in Svedbergunits, which are a measure of the sedimentation velocity of a moleculeunder particular conditions. The determination of the sedimentationvelocity was classically performed in an analytical ultracentrifuge, butis typically now performed in a standard ultracentrifuge. See,Freifelder (1982) Physical Biochemistry (2d ed.) W.H. Freeman & Co., SanFrancisco, Calif.; and Cantor and Schimmel (1980) Biophysical Chemistryparts 1-3, W.H. Freeman & Co., San Francisco, Calif. As a crudedetermination, a sample containing a putatively soluble polypeptide isspun in a standard full sized ultracentrifuge at about 50K rpm for about10 minutes, and soluble molecules will remain in the supernatant. Asoluble particle or polypeptide will typically be less than about 30S,more typically less than about 15S, usually less than about 10S, moreusually less than about 6S, and, in particular embodiments, preferablyless than about 4S, and more preferably less than about 3S. Solubilityof a polypeptide or fragment depends upon the environment and thepolypeptide. Many parameters affect polypeptide solubility, includingtemperature, electrolyte environment, size and molecular characteristicsof the polypeptide, and nature of the solvent. Typically, thetemperature at which the polypeptide is used ranges from about 4° C. toabout 65° C. Usually the temperature at use is greater than about 18° C.and more usually greater than about 22° C.; For diagnostic purposes, thetemperature will usually be about room temperature or warmer, but lessthan the denaturation temperature of components in the assay. Fortherapeutic purposes, the temperature will usually be body temperature,typically about 37° C. for humans, though under certain situations thetemperature may be raised or lowered in situ or in vitro.

[0043] The size and structure of the polypeptide should generally be ina substantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents in a manner which approximates natural lipid bilayerinteractions.

[0044] The solvent will usually be a biologically compatible buffer, ofa type used for preservation of biological activities, and will usuallyapproximate a physiological solvent. Usually the solvent will have aneutral pH, typically between about 5 and 10, and preferably about 7.5.On some occasions, a detergent will be added, typically a mildnon-denaturing one, e.g., CHS or CHAPS, or a low enough concentration asto avoid significant disruption of structural or physiologicalproperties of the protein.

[0045] “Substantially pure” typically means that the protein is isolatedfrom other contaminating proteins, nucleic acids, and other biologicalsderived from the original source organism. Purity, or “isolation” may beassayed by standard methods, and will ordinarily be at least about 50%pure, more ordinarily at least about 60% pure, generally at least about70% pure, more generally at least about 80% pure, often at least about85% pure, more often at least about 90% pure, preferably at least about95% pure, more preferably at least about 98% pure, and in most preferredembodiments, at least 99% pure.

[0046] “Substantial similarity” in the nucleic acid sequence comparisoncontext means either that the segments, or their complementary strands,when compared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 50% of thenucleotides, generally at least 56%, more generally at least 59%,ordinarily at least 62%, more ordinarily at least 65%, often at least68%, more often at least 71%, typically at least 74%, more typically atleast 77%, usually at least 80%, more usually at least about 85%,preferably at least about 90%, more preferably at least about 95 to 98%or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides. Alternatively, substantial similarity exists when thesegments will hybridize under selective hybridization conditions, to astrand, or its complement, typically using a sequence derived from SEQID NO: 1 or 3; 5, 7, or 9; or 11, 13, 15, 17, 19, or 21. Typically,selective hybridization will occur when there is at least about 55%similarity over a stretch of at least about 30 nucleotides, preferablyat least about 65% over a stretch of at least about 25 nucleotides, morepreferably at least about 75%, and most preferably at least about 90%over about 20 nucleotides. See, e.g., Kanehisa (1984) Nucl. Acids Res.12:203-213. The length of similarity comparison, as described, may beover longer stretches, and in certain embodiments will be over a stretchof at least about 17 nucleotides, usually at least about 20 nucleotides,more usually at least about 24 nucleotides, typically at least about 28nucleotides, more typically at least about 40 nucleotides, preferably atleast about 50 nucleotides, and more preferably at least about 75 to 100or more nucleotides.

[0047] “Stringent conditions”, in referring to homology or substantialsimilarity in the hybridization context, will be stringent combinedconditions of salt, temperature, organic solvents, and other parameters,typically those controlled in hybridization reactions. The combinationof parameters is more important than the measure of any singleparameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.31:349-370. A nucleic acid probe which binds to a target nucleic acidunder stringent conditions is specific for said target nucleic acid.Such a probe is typically more than 11 nucleotides in length, and issufficiently identical or complementary to a target nucleic acid overthe region specified by the sequence of the probe to bind the targetunder stringent hybridization conditions.

[0048] Counterpart monocyte proteins from other mammalian species can becloned and isolated by cross-species hybridization of closely relatedspecies. See, e.g., below. Similarity may be relatively low betweendistantly related species, and thus hybridization of relatively closelyrelated species is advisable. Alternatively, preparation of an antibodypreparation which exhibits less species specificity may be useful inexpression cloning approaches.

[0049] The phrase “specifically binds to an antibody” or “specificallyimmunoreactive with”, when referring to a protein or peptide, refers toa binding reaction which is determinative of the presence of the proteinin the presence of a heterogeneous population of proteins and otherbiological components. Thus, under designated immunoassay conditions,the specified antibodies bind to a particular protein and do notsignificantly bind other proteins present in the sample. Specificbinding to an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. Forexample, antibodies raised to the human monocyte protein immunogen withthe amino acid sequence depicted in SEQ ID NO: 2 can be selected toobtain antibodies specifically immunoreactive with that monocyte proteinand not with other proteins. These antibodies recognize proteins highlysimilar to the homologous human monocyte protein.

[0050] III. Nucleic Acids

[0051] These monocyte genes are specifically expressed on dendriticcells. The preferred embodiments, as disclosed, will be useful instandard procedures to isolate genes from other species, e.g., warmblooded animals, such as birds and mammals. Cross hybridization willallow isolation of related proteins from individuals, strains, orspecies. A number of different approaches are available successfully toisolate a suitable nucleic acid clone based upon the informationprovided herein. Southern blot hybridization studies should identifyhomologous genes in other species under appropriate hybridizationconditions.

[0052] Purified protein or defined peptides, are useful for generatingantibodies by standard methods, as described below. Synthetic peptidesor purified protein can be presented to an immune system to generatepolyclonal and monoclonal antibodies. See, e.g., Coligan (1991) CurrentProtocols in Immunology Wiley/Greene, NY; and Harlow and Lane (1989)Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY, which areincorporated herein by reference. Alternatively, a CD protein bindingcomposition can be useful as a specific binding reagent, and advantagecan be taken of its specificity of binding, for, e.g., purification of amonocyte protein.

[0053] The specific binding composition can be used for screening anexpression library made from a cell line which expresses the respectivemonocyte protein. Many methods for screening are available, e.g.,standard staining of surface expressed ligand, or by panning. Screeningof intracellular expression can also be performed by various staining orimmunofluorescence procedures. The binding compositions could be used toaffinity purify or sort out cells expressing the ligand. TABLE 1Sequence encoding a human FDF03 protein, containing Ig domains. Theputative coding region runs from about 154 to 1062. See SEQ ID NO: 1 and2. This 1249 bp clone was isolated from a monocyte cell library. Aputative (hydrophobic) signal sequence runs from −19 to about −1; aputative transmembrane (hydrophobic) segment runs from about 178 to 199.The extracellular region is probably about 170 amino acids, with apotential Ig-like domain structure; the intracellular region is about 80residues. Sequence analysis indicates similarity to GenBank clonesH26010 and R50327 from humans. GTTTGGGGAA GGCTCCTGGC CCCCACAGCCCTCTTCGGAG CCTGAGCCCG GCTCTCCTCA 60 CTCACCTCAA CCCCCAGGCG GCCCCTCCACAGGGCCCCTC TCCTGCCTGG ACGGCTCTGC 120 TGGTCTCCCC GTCCCCTGGA GAAGAACAAGGCC ATG GGT CGG CCC CTG CTG CTG 174                                     Met Gly Arg Pro Leu Leu Leu                                     −19             −15 CCC CTA CTG CCCCTG CTG CTG CCG CCA GCA TTT CTG CAG CCT AGT GGC 222 Pro Leu Leu Pro LeuLeu Leu Pro Pro Ala Phe Leu Gln Pro Ser Gly        −10                  −5                   1 TCC ACA GGA TCT GGTCCA AGC TAC CTT TAT GGG GTC ACT CAA CCA AAA 270 Ser Thr Gly Ser Gly ProSer Tyr Leu Tyr Gly Val Thr Gln Pro Lys  5                  10                  15                  20 CAC CTCTCA GCC TCC ATG GGT GGC TCT GTG GAA ATC CCC TTC TCC TTC 318 His Leu SerAla Ser Met Gly Gly Ser Val Glu Ile Pro Phe Ser Phe                 25                  30                  35 TAT TAC CCCTGG GAG TTA GCC ACA GCT CCC GAC GTG AGA ATA TCC TGG 366 Tyr Tyr Pro TrpGlu Leu Ala Thr Ala Pro Asp Val Arg Ile Ser Trp             40                  45                  50 AGA CGG GGC CACTTC CAC GGG CAG TCC TTC TAC AGC ACA AGG CCG CCT 414 Arg Arg Gly His PheHis Gly Gln Ser Phe Tyr Ser Thr Arg Pro Pro         55                  60                  65 TCC ATT CAC AAG GATTAT GTG AAC CGG CTC TTT CTG AAC TGG ACA GAG 462 Ser Ile His Lys Asp TyrVal Asn Arg Leu Phe Leu Asn Trp Thr Glu     70                  75                  80 GGT CAG AAG AGC GGC TTCCTC AGG ATC TCC AAC CTG CAG AAG CAG GAC 510 Gly Gln Lys Ser Gly Phe LeuArg Ile Ser Asn Leu Gln Lys Gln Asp 85                  90                  95                 100 CAG TCTGTG TAT TTC TGC CGA GTT GAG CTG GAC ACA CGG AGC TCA GGG 558 Gln Ser ValTyr Phe Cys Arg Val Glu Leu Asp Thr Arg Ser Ser Gly                105                 110                 115 AGG CAG CAGTGG CAG TCC ATC GAG GGG ACC AAA CTC TCC ATC ACC CAG 606 Arg Gln Gln TrpGln Ser Ile Glu Gly Thr Lys Leu Ser Ile Thr Gln            120                 125                 130 GCT GTC ACG ACCACC ACC CAG AGG CCC AGC AGC ATG ACT ACC ACC TGG 654 Ala Val Thr Thr ThrThr Gln Arg Pro Ser Ser Met Thr Thr Thr Trp        135                 140                 145 AGG CTC AGT AGC ACAACC ACC ACA ACC GGC CTC AGG GTC ACA CAG GGC 702 Arg Leu Ser Ser Thr ThrThr Thr Thr Gly Leu Arg Val Thr Gln Gly    150                 155                 160 AAA CGA CGC TCA GAC TCTTGG CAC ATA AGT CTG GAG ACT GCT GTG GGG 750 Lys Arg Arg Ser Asp Ser TrpHis Ile Ser Leu Glu Thr Ala Val Gly165                 170                 175                 180 GTG GCAGTG GCT GTC ACT GTG CTC GGA ATC ATG ATT TTG GGA CTG ATC 798 Val Ala ValAla Val Thr Val Leu Gly Ile Met Ile Leu Gly Leu Ile                185                 190                 195 TGC CTC CTCAGG TGG AGG AGA AGG AAA GGT CAG CAG CGG ACT AAA GCC 846 Cys Leu Leu ArgTrp Arg Arg Arg Lys Gly Gln Gln Arg Thr Lys Ala            200                 205                 210 ACA ACC CCA GCCAGG GAA CCC TTC CAA AAC ACA GAG GAG CCA TAT GAG 894 Thr Thr Pro Ala ArgGlu Pro Phe Gln Asn Thr Glu Glu Pro Tyr Glu        215                 220                 225 AAT ATC AGG AAT GAAGGA CAA AAT ACA GAT CCC AAG CTA AAT CCC AAG 942 Asn Ile Arg Asn Glu GlyGln Asn Thr Asp Pro Lys Leu Asn Pro Lys    230                 235                 240 GAT GAC GGC ATC GTA TATGCT TCC CTT GCC CTC TCC AGC TCC ACC TCA 990 Asp Asp Gly Ile Val Tyr AlaSer Leu Ala Leu Ser Ser Ser Thr Ser245                 250                 255                 260 CCC AGAGCA CCT CCC AGC CAC CGT CCC CTC AAG AGC CCC CAG AAC GAG 1038 Pro Arg AlaPro Pro Ser His Arg Pro Leu Lys Ser Pro Gln Asn Glu                265                 270                 275 ACC CTG TACTCT GTC TTA AAG GCC TAACCAATGG ACAGCCCTCT CAAGACTGAA 1092 Thr Leu TyrSer Val Leu Lys Ala             280 TGGTGAGGCC AGGTACAGTG GCGCACACCTGTAATCCCAG CTACTCTGAA GCCTGAGGCA 1152 GAATCAAGTG AGCCCAGGAG TTCAGGGCCAGCTTTGATAA TGGAGCGAGA TGCCATCTCT 1212 AGTTAAAAAT ATATATTAAC AATAAAGTAACAAATTT 1249 A mouse counterpart partial sequence is (SEQ ID NO: 3 and4): CCCCAGTGTC CCTAGACAGA GCATCCTTGC CTTCCTGATG GCTTTGCTGA TCTCGCTTCC 60CTGGAGGGAC TCCAGCC ATG GCT CAG GTC CTG CTT CTG CTC TCA TCA GGC 110                   Met Ala Gln Val Leu Leu Leu Leu Ser Ser Gly                     1               5                  10 TGT CTG CATGCT GGA AAT TCA GAA AGA TAC AAC AGA AAA AAT GGC TTT 158 Cys Leu His AlaGly Asn Ser Glu Arg Tyr Asn Arg Lys Asn Gly Phe             15                  20                  25 GGG GTC AAC CAACCT GAA CGC TGC TCT GGA GTC CAG GGT GGC TCC ATC 206 Gly Val Asn Gln ProGlu Arg Cys Ser Gly Val Gln Gly Gly Ser Ile         30                  35                  40 GAC ATC CCC TTC TCCTTC TAT TTC CCC TGG AAG TTG GCC AAG GAT CCA 254 Asp Ile Pro Phe Ser PheTyr Phe Pro Trp Lys Leu Ala Lys Asp Pro     45                  50                  55 CAG ATG AGC ATA GCC TGGAAA TGG AAG GAT TTC CAT GGG GAA GTC ATC 302 Gln Met Ser Ile Ala Trp LysTrp Lys Asp Phe His Gly Glu Val Ile 60                  65                  70                  75 TAC AACTCC TCC CTG CCT TTC ATA CAT GAG CAC TTC AAG GGC CGG CTC 350 Tyr Asn SerSer Leu Pro Phe Ile His Glu His Phe Lys Gly Arg Leu                 80                  85                  90 ATC CTG AACTGG ACA CAG GGT CAG AC 376 Ile Leu Asn Trp Thr Gln Gly Gln             95 partial human/mouse alignment: huMGRPLLLPLLPLLLPPAFLQPSGSTGSGPSYLYGVTQPKHLSASMGGSVEIPFSFYYPWE moMAQVLLLLSSGCLHAGNSERYNRKNG------FGVNQPERCSGVQGGSIDIPFSFYFPWK huLATAPDVRISWRRGHFHGQSFYSTRPPSIHKDYVNRLFLNWTEGQKSGFLRISNLQK... moLAKDPQMSIAWKWKDFHGEVIYNSSLPFIHEHFKGRLILNWTQGQ...

[0054] TABLE 2 Sequence encoding a protein related to Ig family members,designated YE01, isolated from an activated monocyte cell library. SeeSEQ ID NO: 5 and 6. Signal sequence is indicated. Nucleotide 1247 may beC or T. Sequence analysis suggests YE01 is a member of the Igsuperfamily of receptors, and is closely related to the CD8 family,which contain a V1J-type fold, particularly the Fc receptors alphaand/or gamma. Because it contains an ITAM-like motif, the protein maywell be a monocyte version of the KIR proteins, the Killer InhibitoryReceptors, which send a negative signal to inhibit killer cell function.This protein may share similar function in inhibiting monocyte effectorfunction, e.g., antigen presentation or subsequent response initiation.A mouse counterpart is probably encoded in the EST W55567. ACCGGTCCGGAATTCCCGGG TCGACCCACG CGTCCGGGAA GCCCCATAGG CAGGAGGCCC 60 CCGGGCAGCACATCCTGTCT GCTTGTGTCT GCTGCAGAGT TCTGTCCTTG CATTGGTGCG 120 CCTCAGGCCAGGCTGCACTG CTGGGACCTG GGCC ATG TCT CCC CAC CCC ACC 172                                       Met Ser Pro His Pro Thr                                       −21 −20 GCC CTC CTG GGC CTA GTGCTC TGC CTG GCC CAG ACC ATC CAC ACG CAG 220 Ala Leu Leu Gly Leu Val LeuCys Leu Ala Gln Thr Ile His Thr Gln−15                 −10                  −5                   1 GAG GAAGAT CTG CCC AGA CCC TCC ATC TCG GCT GAG CCA GGC ACC GTG 268 Glu Glu AspLeu Pro Arg Pro Ser Ile Ser Ala Glu Pro Gly Thr Val              5                  10                  15 ATC CCC CTG GGGAGC CAT GTG ACT TTC GTG TGC CGG GGC CCG GTT GGG 316 Ile Pro Leu Gly SerHis Val Thr Phe Val Cys Arg Gly Pro Val Gly         20                  25                  30 GTT CAA ACA TTC CGCCTG GAG AGG GAG AGT AGA TCC ACA TAC AAT GAT 364 Val Gln Thr Phe Arg LeuGlu Arg Glu Ser Arg Ser Thr Tyr Asn Asp     35                  40                  45 ACT GAA GAT GTG TCT CAAGCT AGT CCA TCT GAG TCA GAG GCC AGA TTC 412 Thr Glu Asp Val Ser Gln AlaSer Pro Ser Glu Ser Glu Ala Arg Phe 50                  55                  60                  65 CGC ATTGAC TCA GTA AGT GAA GGA AAT GCC GGG CCT TAT CGC TGC ATC 460 Arg Ile AspSer Val Ser Glu Gly Asn Ala Gly Pro Tyr Arg Cys Ile                 70                  75                  80 TAT TAT AAGCCC CCT AAA TGG TCT GAG CAG AGT GAC TAC CTG GAG CTG 508 Tyr Tyr Lys ProPro Lys Trp Ser Glu Gln Ser Asp Tyr Leu Glu Leu             85                  90                  95 CTG GTG AAA GAAACC TCT GGA GGC CCG GAC TCC CCG GAC ACA GAG CCC 556 Leu Val Lys Glu ThrSer Gly Gly Pro Asp Ser Pro Asp Thr Glu Pro        100                 105                 110 GGC TCC TCA GCT GGACCC ACG CAG AGG CCC TCG GAC AAC AGT CAC AAT 604 Gly Ser Ser Ala Gly ProThr Gln Arg Pro Ser Asp Asn Ser His Asn    115                 120                 125 GAG CAT GCA CCT GCT TCCCAA GGC CTG AAA GCT GAG CAT CTG TAT ATT 652 Glu His Ala Pro Ala Ser GlnGly Leu Lys Ala Glu His Leu Tyr Ile130                 135                 140                 145 CTC ATCGGG GTC TCA GTG GTC TTC CTC TTC TGT CTC CTC CTC CTG GTC 700 Leu Ile GlyVal Ser Val Val Phe Leu Phe Cys Leu Leu Leu Leu Val                150                 155                 160 CTC TTC TGCCTC CAT CGC CAG AAT CAG ATA AAG CAG GGG CCC CCC AGA 748 Leu Phe Cys LeuHis Arg Gln Asn Gln Ile Lys Gln Gly Pro Pro Arg            165                 170                 175 AGC AAG GAC GAGGAG CAG AAG CCA CAG CAG AGG CCT GAC CTG GCT GTT 796 Ser Lys Asp Glu GluGln Lys Pro Gln Gln Arg Pro Asp Leu Ala Val        180                 185                 190 GAT GTT CTA GAG AGGACA GCA GAC AAG GCC ACA GTC AAT GGA CTT CCT 844 Asp Val Leu Glu Arg ThrAla Asp Lys Ala Thr Val Asn Gly Leu Pro    195                 200                 205 GAG AAG GAC AGA GAG ACGGAC ACC TCG GCC CTG GCT GCA GGG AGT TCC 892 Glu Lys Asp Arg Glu Thr AspThr Ser Ala Leu Ala Ala Gly Ser Ser210                 215                 220                 225 CAG GAGGTG ACG TAT GCT CAG CTG GAC CAC TGG GCC CTC ACA CAG AGG 940 Gln Glu ValThr Tyr Ala Gln Leu Asp His Trp Ala Leu Thr Gln Arg                230                 235                 240 ACA GCC CGGGCT GTG TCC CCA CAG TCC ACA AAG CCC ATG GCC GAG TCC 988 Thr Ala Arg AlaVal Ser Pro Gln Ser Thr Lys Pro Met Ala Glu Ser            245                 250                 255 ATC ACG TAT GCAGCC GTT GCC AGA CAC TGACCCCATA CCCACCTGGC 1035 Ile Thr Tyr Ala Ala ValAla Arg His         260                 265 CTCTGCACCT GAGGGTAGAAAGTCACTCTA GGAAAAGCCT GAAGCAGCCA TTTGGAAGGC 1095 TTCCTGTTGG ATTCCTCTTCATCTAGAAAG CCAGCCAGGC AGCTGTCCTG GAGACAAGAG 1155 CTGGAGACTG GAGGTTTCTAACCAGCATCC AGAAGGTTCG TTAGCCAGGT GGTCCCTTCT 1215 ACAATCGGAC AGCTCCTTGGACAGACTGTT TCTCAGTTAT TTCCAAAAAC CCAGCTACAG 1275 TTCC 1279 A similargene was cloned by expressing cloning using a monoclonal antibody DX26,which was raised against the immunogen of human NK cell clone NK681.D5,and selected for inhibiting killing by NK cell clones of Fc receptorbearing target cells (SP2/0). SEQ ID NO: 7 and 8. AAAGGCTGCA GAGTTCTGTCCTTGCATTGG TGCGCCTCAG GCCAGGCTGC ACTGCTGGGA 60 CCTGGGCG ATG TCT CCC CACCCC ACC GCC CTC CTG GGC CTA GTG CTC TGC 110          Met Ser Pro His ProThr Ala Leu Leu Gly Leu Val Leu Cys          −21−20                 −15                 −10 CTG GCC CAG ACC ATC CAC ACGCAG GAG GAA GAT CTG CCC AGA CCC TCC 158 Leu Ala Gln Thr Ile His Thr GlnGlu Glu Asp Leu Pro Arg Pro Ser         −5                   1               5 ATC TCG GCT GAG CCA GGCACC GTG ATC CCC CTG GGG AGC CAT GTG ACT 206 Ile Ser Ala Glu Pro Gly ThrVal Ile Pro Leu Gly Ser His Val Thr 10                  15                  20                  25 TTC GTGTGC CGG GGC CCG GTT GGG GTT CAA ACA TTC CGC CTG GAG AGG 254 Phe Val CysArg Gly Pro Val Gly Val Gln Thr Phe Arg Leu Glu Arg                 30                  35                  40 GAG AGT AGATCC ACA TAC AAT GAT ACT GAA GAT GTG TCT CAA GCT AGT 302 Glu Ser Arg SerThr Tyr Asn Asp Thr Glu Asp Val Ser Gln Ala Ser             45                  50                  55 CCA TCT GAG TCAGAG GCC AGA TTC CGC ATT GAC TCA GTA AGT GAA GGA 350 Pro Ser Glu Ser GluAla Arg Phe Arg Ile Asp Ser Val Ser Glu Gly         60                  65                  70 AAT GCC GGG CCT TATCGC TGC ATC TAT TAT AAG CCC CCT AAA TGG TCT 398 Asn Ala Gly Pro Tyr ArgCys Ile Tyr Tyr Lys Pro Pro Lys Trp Ser     75                  80                  85 GAG CAG AGT GAC TAC CTGGAG CTG CTG GTG AAA GAA ACC TCT GGA GGC 446 Glu Gln Ser Asp Tyr Leu GluLeu Leu Val Lys Glu Thr Ser Gly Gly 90                  95                 100                 105 CCG GACTCC CCG GAC ACA GAG CCC GGC TCC TCA GCT GGA CCC ACG CAG 494 Pro Asp SerPro Asp Thr Glu Pro Gly Ser Ser Ala Gly Pro Thr Gln                110                 115                 120 AGG CCG TCGGAC AAC AGT CAC AAT GAG CAT GCA CCT GCT TCC CAA GGC 542 Arg Pro Ser AspAsn Ser His Asn Glu His Ala Pro Ala Ser Gln Gly            125                 130                 135 CTG AAA GCT GAGCAT CTG TAT ATT CTC ATC GGG GTC TCA GTG GTC TTC 590 Leu Lys Ala Glu HisLeu Tyr Ile Leu Ile Gly Val Ser Val Val Phe        140                 145                 150 CTC TTC TGT CTC CTCCTC CTG GTC CTC TTC TGC CTC CAT CGC CAG AAT 638 Leu Phe Cys Leu Leu LeuLeu Val Leu Phe Cys Leu His Arg Gln Asn    155                 160                 165 CAG ATA AAG CAG GGG CCCCCC AGA AGC AAG GAC GAG GAG CAG AAG CCA 686 Gln Ile Lys Gln Gly Pro ProArg Ser Lys Asp Glu Glu Gln Lys Pro170                 175                 180                 185 CAG CAGAGG CCT GAC CTG GCT GTT GAT GTT CTA GAG AGG ACA GCA GAC 734 Gln Gln ArgPro Asp Leu Ala Val Asp Val Leu Glu Arg Thr Ala Asp                190                 195                 200 AAG GCC ACAGTC AAT GGA CTT CCT GAG AAG GAC AGA GAG ACG GAC ACC 782 Lys Ala Thr ValAsn Gly Leu Pro Glu Lys Asp Arg Glu Thr Asp Thr            205                 210                 215 TCG GCC CTG GCTGCA GGG AGT TCC CAG GAG GTG ACG TAT GCT CAG CTG 830 Ser Ala Leu Ala AlaGly Ser Ser Gln Glu Val Thr Tyr Ala Gln Leu        220                 225                 230 GAC CAC TGG GCC CTCACA CAG AGG ACA GCC CGG GCT GTG TCC CCA CAG 878 Asp His Trp Ala Leu ThrGln Arg Thr Ala Arg Ala Val Ser Pro Gln    235                 240                 245 TCC ACA AAG CCC ATG GCCGAG TCC ATC ACG TAT GCA GCC GTT GCC AGA 926 Ser Thr Lys Pro Met Ala GluSer Ile Thr Tyr Ala Ala Val Ala Arg250                 255                 260                 265 CACTGACCCCATA CCCACCTGGC CTCTGCACCT GAGGGTAGAA AGTCACTCTA 979 HisGGAAAAGCCT GAAGCAGCCA TTTGGAAGGC TTCCTGTTGG ATTCCTCTTC ATCTAGAAAG 1039CCAGCCAGGC AGCTGTCCTG GAGACAAGAG CTGGAGACTG GAGGTTTCTA ACCAGCATCC 1099AGAAGGTTCG TTAGCCAGGT GGTCCCTTCT ACAATCGAGC AGCTCCTTGG ACAGACTGTT 1159TCTCAGTTAT TTCCAGAGAC CCAGCTACAG TTCCCTGGCT GTTTCTAGAG ACCCAGCTTT 1219ATTCACCTGA CTGTTTCCAG AGACCCAGCT AAAGTCACCT GCCTGTTCTA AAGGCCCAGC 1279TACAGCCAAT CAGCCGATTT CCTGAGCAGT GATGCCACCT CCAAGCTTGT CCTAGGTGTC 1339TGCTGTGAAC CTCCAGTGAC CCCAGAGACT TTGCTGTAAT TATCTGCCCT GCTGACCCTA 1399AAGACCTTCC TAGAAGTCAA GAGCTAGCCT TGAGACTGTG CTATACACAC ACAGCTGAGA 1459GCCAAGCCCA GTTCTCTGGG TTGTGCTTTA CTCCACGCAT CAATAAATAA TTTTGAAGGC 1519CTCACATCTG GCAGCCCCAG GCCTGGTCCT GGGTGCATAG GTCTCTCGGA CCCACTCTCT 1579GCCTTCACAG TTGTTCAAAG CTGAGTGAGG GAAACAGGAC TTACGAAAAC GTGTCAGCGT 1639TTTCTTTTTA AAATTTAATT GATCAGGATT GTACGTAAAA AAAAAAAAAA AAAAAAAAAA 1699AAAAAAAAAA AAAAAAAAAA AAAAAAAGG 1728 Nucleic acid and putative aminoacid sequence of soluble DLAIR-2. The signal sequence runs from aboutMet(−21) to Thr(−1) (SEQ ID NO: 9 and 10). CCACGCGTCC GGGGACCGGG GCC ATGTCT CCA CAC CTC ACT GCT CTC CTG 50                           Met Ser ProHis Leu Thr Ala Leu Leu                           −21−20                 −15 GGC CTA GTG CTC TGC CTG GCC CAG ACC ATC CAC ACGCAG GAG GGG GCC 98 Gly Leu Val Leu Cys Leu Ala Gln Thr Ile His Thr GlnGlu Gly Ala         −10                  −5                   1 CTT CCCAGA CCC TCC ATC TCG GCT GAG CCA GGC ACT GTG ATC TCC CCG 146 Leu Pro ArgPro Ser Ile Ser Ala Glu Pro Gly Thr Val Ile Ser Pro  5                  10                  15                  20 GGG AGCCAT GTG ACT TTC ATG TGC CGG GGC CCG GTT GGG GTT CAA ACA 194 Gly Ser HisVal Thr Phe Met Cys Arg Gly Pro Val Gly Val Gln Thr                 25                  30                  35 TTC CGC CTGGAG AGG GAG GAT AGA GCC AAG TAC AAA GAT AGT TAT AAT 242 Phe Arg Leu GluArg Glu Asp Arg Ala Lys Tyr Lys Asp Ser Tyr Asn             40                  45                  50 GTG TTT CGA CTTGGT CCA TCT GAG TCA GAG GCC AGA TTC CAC ATT GAC 290 Val Phe Arg Leu GlyPro Ser Glu Ser Glu Ala Arg Phe His Ile Asp         55                  60                  65 TCA GTA AGT GAA GGAAAT GCC GGG CTT TAT CGC TGC CTC TAT TAT AAG 338 Ser Val Ser Glu Gly AsnAla Gly Leu Tyr Arg Cys Leu Tyr Tyr Lys     70                  75                  80 CCC CCT GGA TGG TCT GAGCAC AGT GAC TTC CTG GAG CTG CTG GTG AAA 386 Pro Pro Gly Trp Ser Glu HisSer Asp Phe Leu Glu Leu Leu Val Lys 85                  90                  95                 100 GGG ACTGTG CCA GGC ACT GAA GCC TCC GGA TTT GAT GCA CCA 428 Gly Thr Val Pro GlyThr Glu Ala Ser Gly Phe Asp Ala Pro                105                 110 TGAATGAGGA GAAATGGCCT CCCGTCTTGTGAACTTCAAT GGGGAGAAAT AATTAGAATG 488 AGCAATAGAA ATGCACAGAT GCCTATACATACATATACAA ATAAAAAGAT ACGATTCGCA 548 AAAAAAAAAA AAAAAAGGGC 568

[0055] TABLE 3 Human KTE03 sequences, e.g., alternative splicing,encoding related proteins with homology to several NK KIR surfacemolecules, and to the Fc receptors gamma and alpha. YYB01 codingsequence appears to run from about 81 to 1397. The message appears to beIL- 10 upregulated. See SEQ ID NO: 11 and 12. Because of significantidentity of sequence which ends at specific locations, it appears thatthere may be splice junctions around nucleotide 36, 1264, and 1587. TheYYB04 sequence provided below indicates that certain insertions ofsequence lead to a frameshift and alternative carboxy terminal sequence.Moreover, certain peculiar differences in sequence suggest eithersequencing errors, or a mechanism of variability generated by amechanism perhaps analogous to hypervariable region combinations.GTCGACCCAC GCGTCCGCCT CTGTCCTGCC AGCACCGAGG GCTCATCCAT CCACAGAGCA 60GTGCAGTGGG AGGAGACGCC ATG ACC CCC ATC CTC ACG GTC CTG ATC TGT 110                      Met Thr Pro Ile Leu Thr Val Leu Ile Cys                        1               5                  10 CTC GGGCTG AGC CTG GAC CCC AGG ACC CAC GTG CAG GCA GGG CCC CTC 158 Leu Gly LeuSer Leu Asp Pro Arg Thr His Val Gln Ala Gly Pro Leu                 15                  20                  25 CCC AAG CCCACC CTC TGG GCT GAG CCA GGC TCT GTG ATC ACC CAA GGG 206 Pro Lys Pro ThrLeu Trp Ala Glu Pro Gly Ser Val Ile Thr Gln Gly             30                  35                  40 AGT CCT GTG ACCCTC AGG TGT CAG GGG AGC CTG GAG ACG CAG GAG TAC 254 Ser Pro Val Thr LeuArg Cys Gln Gly Ser Leu Glu Thr Gln Glu Tyr         45                  50                  55 CAT CTA TAT AGA GAAAAG AAA ACA GCA CTC TGG ATT ACA CGG ATC CCA 302 His Leu Tyr Arg Glu LysLys Thr Ala Leu Trp Ile Thr Arg Ile Pro     60                  65                  70 CAG GAG CTT GTG AAG AAGGGC CAG TTC CCC ATC CTA TCC ATC ACC TGG 350 Gln Glu Leu Val Lys Lys GlyGln Phe Pro Ile Leu Ser Ile Thr Trp 75                  80                  85                  90 GAA CATGCA GGG CGG TAT TGC TGT ATC TAT GGC AGC CAC ACT GCA GGC 398 Glu His AlaGly Arg Tyr Cys Cys Ile Tyr Gly Ser His Thr Ala Gly                 95                 100                 105 CTC TCA GAGAGC AGT GAC CCC CTG GAG CTG GTG GTG ACA GGA GCC TAC 446 Leu Ser Glu SerSer Asp Pro Leu Glu Leu Val Val Thr Gly Ala Tyr            110                 115                 120 AGC AAA CCC ACCCTC TCA GCT CTG CCC AGC CCT GTG GTG ACC TCA GGA 494 Ser Lys Pro Thr LeuSer Ala Leu Pro Ser Pro Val Val Thr Ser Gly        125                 130                 135 GGG AAT GTG ACC ATCCAG TGT GAC TCA CAG GTG GCA TTT GAT GGC TTC 542 Gly Asn Val Thr Ile GlnCys Asp Ser Gln Val Ala Phe Asp Gly Phe    140                 145                 150 ATT CTG TGT AAG GAA GGAGAA GAT GAA CAC CCA CAA TGC CTG AAC TCC 590 Ile Leu Cys Lys Glu Gly GluAsp Glu His Pro Gln Cys Leu Asn Ser155                 160                 165                 170 CAT TCCCAT GCC CGT GGG TCA TCC CGG GCC ATC TTC TCC GTG GGC CCC 638 His Ser HisAla Arg Gly Ser Ser Arg Ala Ile Phe Ser Val Gly Pro                175                 180                 185 GTG AGC CCAAGT CGC AGG TGG TCG TAC AGG TGC TAT GGT TAT GAC TCG 686 Val Ser Pro SerArg Arg Trp Ser Tyr Arg Cys Tyr Gly Tyr Asp Ser            190                 195                 200 CGC GCT CCC TATGTG TGG TCT CTA CCC AGT GAT CTC CTG GGG CTC CTG 734 Arg Ala Pro Tyr ValTrp Ser Leu Pro Ser Asp Leu Leu Gly Leu Leu        205                 210                 215 GTC CCA GGT GTT TCTAAG AAG CCA TCA CTC TCA GTG CAG CCG GGT CCT 782 Val Pro Gly Val Ser LysLys Pro Ser Leu Ser Val Gln Pro Gly Pro    220                 225                 230 GTC GTG GCC CCT GGG GAGAAG CTG ACC TTC CAG TGT GGC TCT GAT GCC 830 Val Val Ala Pro Gly Glu LysLeu Thr Phe Gln Cys Gly Ser Asp Ala235                 240                 245                 250 GGC TACGAC AGA TTT GTT CTG TAC AAG GAG TGG GGA CGT GAC TTC CTC 878 Gly Tyr AspArg Phe Val Leu Tyr Lys Glu Trp Gly Arg Asp Phe Leu                255                 260                 265 CAG CGC CCTGGC CGG CAC CCC CAG GCT GGG CTC TCC CAG GCC AAC TTC 926 Gln Arg Pro GlyArg Gln Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe            270                 275                 280 ACC CTG GGC CCTGTG AGC CGC TCC TAC GGG GGC CAG TAC ACA TGC TCC 974 Thr Leu Gly Pro ValSer Arg Ser Tyr Gly Gly Gln Tyr Thr Cys Ser        285                 290                 295 GGT GCA TAC AAC CTCTCC TCC GAG TGG TCG GCC CCC AGC GAC CCC CTG 1022 Gly Ala Tyr Asn Leu SerSer Glu Trp Ser Ala Pro Ser Asp Pro Leu    300                 305                 310 GAC ATC CTG ATC ACA GGACAG ATC CGT GCC AGA CCC TTC CTC TCC GTG 1070 Asp Ile Leu Ile Thr Gly GlnIle Arg Ala Arg Pro Phe Leu Ser Val315                 320                 325                 330 CGG CCGGGC CCC ACA GTG GCC TCA GGA GAG AAC GTG ACC CTG CTG TGT 1118 Arg Pro GlyPro Thr Val Ala Ser Gly Glu Asn Val Thr Leu Leu Cys                335                 340                 345 CAG TCA CAGGGA GGG ATG CAC ACT TTC CTT TTG ACC AAG GAG GGG GCA 1166 Gln Ser Gln GlyGly Met His Thr Phe Leu Leu Thr Lys Glu Gly Ala            350                 355                 360 GCT GAT TCC CCGCTG CGT CTA AAA TCA AAG CGC CAA TCT CAT AAG TAC 1214 Ala Asp Ser Pro LeuArg Leu Lys Ser Lys Arg Gln Ser His Lys Tyr        365                 370                 375 CAG GCT GAA TTC CCCATG AGT CCT GTG ACC TCG GCC CAC GCG GGG ACC 1262 Gln Ala Glu Phe Pro MetSer Pro Val Thr Ser Ala His Ala Gly Thr    380                 385                 390 TAC AGG TGC TAC GGC TCACTC AGC TGG AAC CCC TAC CTG CTG ACT CAC 1310 Tyr Arg Cys Tyr Gly Ser LeuSer Ser Asn Pro Tyr Leu Leu Thr His395                 400                 405                 410 CCC AGTGAC CCC CTG GAG CTC GTG GTC TCA GGA GCA GCT GAG ACC CTC 1358 Pro Ser AspPro Leu Glu Leu Val Val Ser Gly Ala Ala Glu Thr Leu                415                 420                 425 AGC CCA CCACAA AAC AAG TCC GAC TCC AAG GCT GGT GAG TGAGGAGATG 1407 Ser Pro Pro GlnAsn Lys Ser Asp Ser Lys Ala Gly Glu             430                 435CTTGCCGTGA TGACGGTGGG CACAGAGGGT CAGGTCCTGT CAAGAGGAGC TGGGTGTCCT 1467GGGTGGACAT TTGAAGAATT ATATTCATTC CAACTTGAAG AATTATTCAA CACCTTTAAC 1527AATGTATATG TGAAGTACTT TATTCTTTCA TATTTTAAAA ATAAAAGATA ATTATCCATG 1587AAAAAAAAAA AAAAAAAAAA AAAGGGCGGC CGC 1620 YYB04: Related to YYB01,apparently through alternative splicing from the same or a very highlyrelated gene. The coding region runs from about 191 to 1493, but theinitiation methionine may actually be at the numbered Met at 18. See SEQID NO: 13 and 14. Another transcript was isolated which containsevidence for existence of an insert of sequence TGCTACGGCT CACTCAACTCCGACCCCTAC CTGCTGTCTC ACCCCAGTGA GCCCCTGGAG CTCGTGGTCT CAGG betweenresidues 1426 and 1427, which changes the downstream reading frame ofthe subsequent sequence, to encode, from residue 413, CYG SLNSD PYLLSHPSEP LELVV SGPSM GSSPP PTGPI STPAG PEDQP LTPTG SDPQS GLGRH LGVVI GILVAVVLLL LLLLL LFLIL RHRRQ GKHWT STQRK ADFQH PAGAV GPEPT DRGLQ WRSSP AADAQEENLY AAVKD TQPED GVEMD TRAAA SEAPQ DVTYA QLHSL TLRRK ATEPP PSQER EPPAEPSIYA TLAIH. (SEQ ID NO: 15 and 16.) This alternative sequence containsa transmembrane segment from about 478 to 500. GTCGACCCAC GCGTCCGGTCAACTTTTCTT CCCCTACTTC CCTGCATTTC TCCTCTGTGC 60 TCACTGCCAC ACGCAGCTCAACCTGGACGG CACAGCCAGA TGCGAGATGC GTCTCTGCTG 120 ATCTGAGTCT GCCTGCAGCATGGACCTGGG TCTTCCCTGA AGCATCTCCA GGGCTGGAGG 180 GACGACTGCC ATG CAC CGAGGG CTC ATC CAT CCG CAG AGC AGG GCA GTG 229            Met His Arg GlyLeu Ile His Pro Gln Ser Arg Ala Val             1               5                  10 GGA GGA GAC GCC ATGACC CCC ATC GTC ACA GTC CTG ATC TGT CTC GGG 277 Gly Gly Asp Ala Met ThrPro Ile Val Thr Val Leu Ile Cys Leu Gly     15                  20                  25 CTG AGT CTG GGC CCC AGGACC CAC GTG CAG ACA GGG ACC ATC CCC AAG 325 Leu Ser Leu Gly Pro Arg ThrHis Val Gln Thr Gly Thr Ile Pro Lys 30                  35                  40                  45 CCC ACCCTG TGG GCT GAG CCA GAC TCT GTG ATC ACC CAG GGG AGT CCC 373 Pro Thr LeuTrp Ala Glu Pro Asp Ser Val Ile Thr Gln Gly Ser Pro                 50                  55                  60 GTC ACC CTCAGT TGT CAG GGG AGC CTT GAA GCC CAG GAG TAC CGT CTA 421 Val Thr Leu SerCys Gln Gly Ser Leu Glu Ala Gln Glu Tyr Arg Leu             65                  70                  75 TAT AGG GAG AAAAAA TCA GCA TCT TGG ATT ACA CGG ATA CGA CCA GAG 469 Tyr Arg Glu Lys LysSer Ala Ser Trp Ile Thr Arg Ile Arg Pro Glu         80                  85                  90 CTT GTG AAG AAC GGCCAG TTC CAC ATC CCA TCC ATC ACC TGG GAA CAC 517 Leu Val Lys Asn Gly GlnPhe His Ile Pro Ser Ile Thr Trp Glu His     95                 100                 105 ACA GGG CGA TAT GGC TGTCAG TAT TAC AGC CGC GCT CGG TGG TCT GAG 565 Thr Gly Arg Tyr Gly Cys GlnTyr Tyr Ser Arg Ala Arg Trp Ser Glu110                 115                 120                 125 CTC AGTGAC CCC CTG GTG CTG GTG ATG ACA GGA GCC TAC CCA AAA CCC 613 Leu Ser AspPro Leu Val Leu Val Met Thr Gly Ala Tyr Pro Lys Pro                130                 135                 140 ACC CTC TCAGCC CAG CCC AGC CCT GTG GTG ACC TCA GGA GGA AGG GTG 661 Thr Leu Ser AlaGln Pro Ser Pro Val Val Thr Ser Gly Gly Arg Val            145                 150                 155 ACC CTC CAG TGTGAG TCA CAG GTG GCA TTT GGC GGC TTC ATT CTG TGT 709 Thr Leu Gln Cys GluSer Gln Val Ala Phe Gly Gly Phe Ile Leu Cys        160                 165                 170 AAG GAA GGA GAA GATGAA CAC CCA CAA TGC CTG AAC TCC CAG CCC CAT 757 Lys Glu Gly Glu Asp GluHis Pro Gln Cys Leu Asn Ser Gln Pro His    175                 180                 185 GCC CGT GGG TCG TCC CGCGCC ATC TTC TCC GTG GGC CCC GTG AGC CCG 805 Ala Arg Gly Ser Ser Arg AlaIle Phe Ser Val Gly Pro Val Ser Pro190                 195                 200                 205 AAT CGCAGG TGG TCG CAC AGG TGC TAT GGT TAT GAC TTG AAC TCT CCC 853 Asn Arg ArgTrp Ser His Arg Cys Tyr Gly Tyr Asp Leu Asn Ser Pro                210                 215                 220 TAT GTG TGGTCT TCA CCC AGT GAT CTC CTG GAG CTC CTG GTC CCA GGT 901 Tyr Val Trp SerSer Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly            225                 230                 235 GTT TCT AAG AAGCCA TCA CTC TCA GTG CAG CCG GGT CCT GTC GTG GCC 949 Val Ser Lys Lys ProSer Leu Ser Val Gln Pro Gly Pro Val Val Ala        240                 245                 250 CCT GGG GAA AGC CTGACC CTC CAG TGT GTC TCT GAT GTC GGC TAT GAC 997 Pro Gly Glu Ser Leu ThrLeu Gln Cys Val Ser Asp Val Gly Tyr Asp    255                 260                 265 AGA TTT GTT CTG TAC AAGGAG GGG GAA CGT GAC CTT CGC CAG CTC CCT 1045 Arg Phe Val Leu Tyr Lys GluGly Glu Arg Asp Leu Arg Gln Leu Pro270                 275                 280                 285 GGC CGGCAG CCC CAG GCT GGG CTC TCC CAG GCC AAC TTC ACC CTG GGC 1093 Gly Arg GlnPro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly                290                 295                 300 CCT GTG AGCCGC TCC TAC GGG GGC CAG TAC AGA TGC TAC GGT GCA TAC 1141 Pro Val Ser ArgSer Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala Tyr            305                 310                 315 AAC CTC TCC TCCGAG TGG TCG GCC CCC AGC GAC CCC CTG GAC ATC CTG 1189 Asn Leu Ser Ser GluTrp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu        320                 325                 330 ATC ACA GGA CAG ATCCAT GGC ACA CCC TTC ATC TCA GTG CAG CCA GGC 1237 Ile Thr Gly Gln Ile HisGly Thr Pro Phe Ile Ser Val Gln Pro Gly    335                 340                 345 CCC ACA GTG GCC TCA GGAGAG AAC GTG ACC CTG CTG TGT CAG TCA TGG 1285 Pro Thr Val Ala Ser Gly GluAsn Val Thr Leu Leu Gys Gln Ser Trp350                 355                 360                 365 CGG CAGTTC CAC ACT TTC CTT CTG ACC AAG GCG GGA GCA GCT GAT GCC 1333 Arg Gln PheHis Thr Phe Leu Leu Thr Lys Ala Gly Ala Ala Asp Ala                370                 375                 380 CCA CTC CGTCTA AGA TCA ATA CAC GAA TAT CCT AAG TAC CAG GCT GAA 1381 Pro Leu Arg LeuArg Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala Glu            385                 390                 395 TTC CCC ATG AGTCCT GTG ACC TCA GCC CAC GCG GGG ACC TAC AGG ACC 1429 Phe Pro Met Ser ProVal Thr Ser Ala His Ala Gly Thr Tyr Arg Thr        400                 405                 410 CTC CAT GGG TTC CAGCCC CCC ACC CAC CGG TCC CAT CTC CAC ACC TGC 1477 Leu His Gly Phe Gln ProPro Thr His Arg Ser His Leu His Thr Cys    415                 420                 425 AGG CCC TGAGGACCAGCCCCTCACCC CCACTGGGTC GGATCCCCAA AGTGGTCTGG 1533 Arg Pro 430 GAAGGCACCTGGGGGTTGTG ATCGGCATCT TGGTGGCCGT CGTCCTACTG CTCCTCCTCC 1593 TCCTCCTCCTCTTCCTCATC CTCCGACATC GACGTCAGGG CAAACACTGG ACATCGACCC 1653 AGAGAAAGGCTGATTTCCAA CATCCTGCAG GGGCTGTGGG GCCAGAGCCC ACAGACAGAG 1713 GCCTGCAGTGGAGGTCCAGC CCAGCTGCCG ACGCCCAGGA AGAAAACCTC TATGCTGCCG 1773 TGAAGGACACACAGCCTGAA GATGGGGTGG AGATGGACAC TCGGGCTGCT GGATCTGAAG 1833 CCCCCCAGGATGTGACCTAC GCCCAGCTGC ACAGCTTGAC CCTCAGACGG AAGGCAACTG 1893 AGCCTCCTCCATCCCAGGAA AGGGAACCTC CAGCTGAGCC CAGCATTTAC GCCACCCTGG 1953 CCATCCACTAGCCCGGAGGG TACGCAGACT CCACACTCAG TAGAAGGAGA CTCAGGACTG 2013 CTGAAGGCACGGGAGCTGCC CCCAGTGGAC ACCAATGAAC CCCAGTCAGC CTGGACCCCT 2073 AACAAAGACCATGAGGAGAT GCTGGGAACT TTGGGACTCA CTTGATTCTG CAGTGGAAAT 2133 AACTAATATCCCTACATTTT TTAATTAAAG CAACAGACTT CTCAATAATC AATGAGTTAA 2193 CCGA 2197 AKTE03 embodiment designated KLM63 (SEQ ID NO: 17 and 18): AAAGAAGTCAACTTTTCTTC CCCTACTTCC CTGCATTTCT CCTCTGTGCT CACTGCCACA 60 CGCAGCTCAACCTGGACGGC ACAGCCAGAT GCGAGATGCG TCTCTGCTGA TCTGAGTCTG 120 CCTGCAGCATGGACCTGGGT CTTCCCTGAA GCATCTCCAG GGCTGGAGGG ACGACTGCC 179 ATG CAC CGAGGG CTC ATC CAT CCG CAG AGC AGG GCA GTG GGA GGA GAC 227 Met His Arg GlyLeu Ile His Pro Gln Ser Arg Ala Val Gly Gly Asp  1               5                  10                  15 GCC ATG ACCCCC ATC GTC ACA GTC CTG ATC TGT CTC GGG CTG AGT CTG 275 Ala Met Thr ProIle Val Thr Val Leu Ile Cys Leu Gly Leu Ser Leu             20                  25                  30 GGC CCC AGG ACCCAC GTG CAG ACA GGG ACC ATC CCC AAG CCC ACC CTG 323 Gly Pro Arg Thr HisVal Gln Thr Gly Thr Ile Pro Lys Pro Thr Leu         35                  40                  45 TGG GCT GAG CCA GACTCT GTG ATC ACC CAG GGG AGT CCC GTC ACC CTC 371 Trp Ala Glu Pro Asp SerVal Ile Thr Gln Gly Ser Pro Val Thr Leu     50                  55                  60 AGT TGT CAG GGG AGC CTTGAA GCC CAG GAG TAC CGT CTA TAT AGG GAG 419 Ser Cys Gln Gly Ser Leu GluAla Gln Glu Tyr Arg Leu Tyr Arg Glu 65                  70                  75                  80 AAA AAATCA GCA TCT TGG ATT ACA CGG ATA CGA CCA GAG CTT GTG AAG 467 Lys Lys SerAla Ser Trp Ile Thr Arg Ile Arg Pro Glu Leu Val Lys                 85                  90                  95 AAC GGC CAGTTC CAC ATC CCA TCC ATC ACC TGG GAA CAC ACA GGG CGA 515 Asn Gly Gln PheHis Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg            100                 105                 110 TAT GGC TGT CAGTAT TAC AGC CGC GCT CGG TGG TCT GAG CTC AGT GAC 563 Tyr Gly Cys Gln TyrTyr Ser Arg Ala Arg Trp Ser Glu Leu Ser Asp        115                 120                 125 CCC CTG GTG CTG GTGATG ACA GGA GCC TAC CCA AAA CCC ACC CTC TCA 611 Pro Leu Val Leu Val MetThr Gly Ala Tyr Pro Lys Pro Thr Leu Ser    130                 135                 140 GCC CAG CCC AGC CCT GTGGTG ACC TCA GGA GGA AGG GTG ACC CTC CAG 659 Ala Gln Pro Ser Pro Val ValThr Ser Gly Gly Arg Val Thr Leu Gln145                 150                 155                 160 TGT GAGTCA CAG GTG GCA TTT GGC GGC TTC ATT CTG TGT AAG GAA GGA 707 Cys Glu SerGln Val Ala Phe Gly Gly Phe Ile Leu Cys Lys Glu Gly                165                 170                 175 GAA GAT GAACAC CCA CAA TGG CTG AAC TCC CAG CCC CAT GCC CGT GGG 755 Glu Asp Glu HisPro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly            180                 185                 190 TCG TCC CGC GCCATC TTC TCC GTG GGC CCC GTG AGC CCG AAT CGC AGG 803 Ser Ser Arg Ala IlePhe Ser Val Gly Pro Val Ser Pro Asn Arg Arg        195                 200                 205 TGG TCG CAC AGG TGCTAT GGT TAT GAC TTG AAC TCT CCC TAT GTG TGG 851 Trp Ser His Arg Cys TyrGly Tyr Asp Leu Asn Ser Pro Tyr Val Trp    210                 215                 220 TCT TCA CCC AGT GAT CTCCTG GAG CTC CTG GTC CCA GGT GTT TCT AAG 899 Ser Ser Pro Ser Asp Leu LeuGlu Leu Leu Val Pro Gly Val Ser Lys225                 230                 235                 240 AAG CCATCA CTC TCA GTG CAG CCG GGT CCT GTC GTG GCC CCT GGG GAA 947 Lys Pro SerLeu Ser Val Gln Pro Gly Pro Val Val Ala Pro Gly Glu                245                 250                 255 AGC CTG ACCCTC CAG TGT GTC TCT GAT GTC GGC TAT GAC AGA TTT GTT 995 Ser Leu Thr LeuGln Cys Val Ser Asp Val Gly Tyr Asp Arg Phe Val            260                 265                 270 CTG TAC AAG GAGGGG GAA CGT GAC CTT CGC CAG CTC CCT GGC CGG CAG 1043 Leu Tyr Lys Glu GlyGlu Arg Asp Leu Arg Gln Leu Pro Gly Arg Gln        275                 280                 285 CCC CAG GCT GGG CTCTCC CAG GCC AAC TTC ACC CTG GGC CCT GTG AGC 1091 Pro Gln Ala Gly Leu SerGln Ala Asn Phe Thr Leu Gly Pro Val Ser    290                 295                 300 CGC TCC TAG GGG GGC CAGTAC AGA TGC TAC GGT GCA TAC AAC CTC TCC 1139 Arg Ser Tyr Gly Gly Gln TyrArg Cys Tyr Gly Ala Tyr Asn Leu Ser305                 310                 315                 320 TCC GAGTGG TCG GCC CCC AGC GAC CCC CTG GAC ATC CTG ATC ACA GGA 1187 Ser Glu TrpSer Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly                325                 330                 335 CAG ATC CATGGC ACA CCC TTC ATC TCA GTG CAG CCA GGC CCC ACA GTG 1235 Gln Ile His GlyThr Pro Phe Ile Ser Val Gln Pro Gly Pro Thr Val            340                 345                 350 GCC TCA GGA GAGAAC GTG ACC CTG CTG TGT CAG TCA TGG CGG CAG TTC 1283 Ala Ser Gly Glu AsnVal Thr Leu Leu Cys Gln Ser Trp Arg Gln Phe        355                 360                 365 CAC ACT TTC CTT CTGACC AAG GCG GGA GCA GCT GAT GCC CCA CTC CGT 1331 His Thr Phe Leu Leu ThrLys Ala Gly Ala Ala Asp Ala Pro Leu Arg    370                 375                 380 CTA AGA TCA ATA CAC GAATAT CCT AAG TAC CAG GCT GAA TTC CCC ATG 1379 Leu Arg Ser Ile His Glu TyrPro Lys Tyr Gln Ala Glu Phe Pro Met385                 390                 395                 400 AGT CCCGTG ACC TCA GCC CAC GCG GGG ACC TAC AGG TGC TAC GGC TCA 1427 Ser Pro ValThr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser                405                 410                 415 CTC AAC TCCGAC CCC TAC CTG CTG TCT CAC CCC AGT GAG CCC CTG GAG 1475 Leu Asn Ser AspPro Tyr Leu Leu Ser His Pro Ser Glu Pro Leu Glu            420                 425                 430 CTC GTG GTC TCAGGA CCC TCC ATG GGT TCC AGC CCC CCA CCC ACC GGT 1523 Leu Val Val Ser GlyPro Ser Met Gly Ser Ser Pro Pro Pro Thr Gly        435                 440                 445 CCC ATC TCC ACA CCTGCA GGC CCT GAG GAC CAG CCC CTC ACC CCC ACT 1571 Pro Ile Ser Thr Pro AlaGly Pro Glu Asp Gln Pro Leu Thr Pro Thr    450                 455                 460 GGG TCG GAT CCC CAA AGTGGT CTG GGA AGG CAC CTG GGG GTT GTG ATC 1619 Gly Ser Asp Pro Gln Ser GlyLeu Gly Arg His Leu Gly Val Val Ile465                 470                 475                 480 GGC ATCTTG GTG GCC GTC GTC CTA CTG CTC CTC CTC CTC CTC CTC CTC 1667 Gly Ile LeuVal Ala Val Val Leu Leu Leu Leu Leu Leu Leu Leu Leu                485                 490                 495 TTC CTC ATCCTC CGA CAT CGA CGT CAG GGC AAA CAC TGG ACA TCG ACC 1715 Phe Leu Ile LeuArg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr            500                 505                 510 CAG AGA AAG GCTGAT TTC CAA CAT CCT GCA CCC GCT GTG GGG CCA GAG 1763 Gln Arg Lys Ala AspPhe Gln His Pro Ala Gly Ala Val Gly Pro Glu        515                 520                 525 CCC ACA GAC AGA GGCCTG CAG TGG AGG TCC AGC CCA GCT GCC GAC GCC 1811 Pro Thr Asp Arg Gly LeuGln Trp Arg Ser Ser Pro Ala Ala Asp Ala    530                 535                 540 CAG GAA GAA AAC CTC TATGCT GCC GTG AAG GAC ACA CAG CCT GAA GAT 1859 Gln Glu Glu Asn Leu Tyr AlaAla Val Lys Asp Thr Gln Pro Glu Asp545                 550                 555                 560 GGG GTGGAG ATG GAC ACT CGG GCT GCT GCA TCT GAA GCC CCC CAG GAT 1907 Gly Val GluMet Asp Thr Arg Ala Ala Ala Ser Glu Ala Pro Gln Asp                565                 570                 575 GTG ACC TACGCC CAG CTG CAC AGC TTG ACC CTC AGA CGG AAG GCA ACT 1955 Val Thr Tyr AlaGln Leu His Ser Leu Thr Leu Arg Arg Lys Ala Thr            580                 585                 590 GAG CCT CCT CCATCC CAG GAA AGG GAA CCT CCA GCT GAG CCC AGC ATC 2003 Glu Pro Pro Pro SerGln Glu Arg Glu Pro Pro Ala Glu Pro Ser Ile        595                 600                 605 TAC GCC ACC CTG GCCATC CAC TAGCCCGGAG GGTACGCAGA CTCCACACTC 2054 Tyr Ala Thr Leu Ala IleHis     610                 615 AGTAGAAGGA GACTCAGGAC TGCTGAAGGCACGGGAGCTG CCCCCAGTGG ACACCAATGA 2114 ACCCCAGTCA GCCTGGACCC CTAACAAAGACCATGAGGAG ATGCTGGGAA CTTTGGGACT 2174 CACTTGATTC TGCAGTCGAA ATAACTAATATCCCTACATT TTTTAATTAA AGCAACAGAC 2234 TTCTCAATAA TCAATGAGTT AACCGAGAAAACTAAAATCA GAAGTAAGAA TGTGCTTTAA 2294 ACTGAATCAC AATATAAATA TTACACATCACACAATGAAA TTGAAAAAGT ACAAACCACA 2354 AATGAAAAAA GTAGAAACGA AAAAAAAAAAAAAA 2388 A KTE03 embodiment designated KLM66 (SEQ IDS NO: 19 and 20):GTCAACTTTT CTTCCCCTAC TTCCCTGCAT TTCTCCTCTG TGCTCACTGC CACACGCAGC 60TCAACCTGGA CGGCACAGCC AGATGCGAGA TGCGTCTCTG CTGATCTGAG TCTGCCTGCA 120GCATGGACCT GGGTCTTCCC TGAAGCATCT CCAGGGCTGG AGGGACGACT GCC ATG 176                                                           Met                                                             1 CAC CGAGGG CTC ATC CAT CCG CAG AGC AGG GCA GTG GGA GGA GAC GCC 224 His Arg GlyLeu Ile His Pro Gln Ser Arg Ala Val Gly Gly Asp Ala              5                  10                  15 ATG ACC CCC ATCGTC ACA GTC CTG ATC TGT CTC GGG CTG AGT CTG GGC 272 Met Thr Pro Ile ValThr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly         20                  25                  30 CCC AGG ACC CAC GTGCAG ACA GGG ACC ATC CCC AAG CCC ACC CTG TGG 320 Pro Arg Thr His Val GlnThr Gly Thr Ile Pro Lys Pro Thr Leu Trp     35                  40                  45 GCT GAG CCA GAC TCT GTGATC ACC CAG GGG AGT CCC GTC ACC CTC AGT 368 Ala Glu Pro Asp Ser Val IleThr Gln Gly Ser Pro Val Thr Leu Ser 50                  55                  60                  65 TGT CAGGGG AGC CTT GAA GCC CAG GAG TAC CGT CTA TAT AGG GAG AAA 416 Cys Gln GlySer Leu Glu Ala Gln Glu Tyr Arg Leu Tyr Arg Glu Lys                 70                  75                  80 AAA TCA GCATCT TGG ATT ACA CGG ATA CGA CCA GAG CTT GTG AAG AAC 464 Lys Ser Ala SerTrp Ile Thr Arg Ile Arg Pro Glu Leu Val Lys Asn             85                  90                  95 GGC CAG TTC CACATC CCA TCC ATC ACC TGG GAA CAC ACA GGG CGA TAT 512 Gly Gln Phe His IlePro Ser Ile Thr Trp Glu His Thr Gly Arg Tyr        100                 105                 110 GGC TGT CAG TAT TACAGC CGC GCT CGG TGG TCT GAG CTC AGT GAC CCC 560 Gly Cys Gln Tyr Tyr SerArg Ala Arg Trp Ser Glu Leu Ser Asp Pro    115                 120                 125 CTG GTG CTG GTG ATG ACAGGA GCC TAC CCA AAA CCC ACC CTC TCA GCC 608 Leu Val Leu Val Met Thr GlyAla Tyr Pro Lys Pro Thr Leu Ser Ala130                 135                 140                 145 CAG CCCAGC CCT GTG GTG ACC TCA GGA GGA AGG GTG ACC CTC CAG TGT 656 Gln Pro SerPro Val Val Thr Ser Gly Gly Arg Val Thr Leu Gln Cys                150                 155                 160 GAG TCA CAGGTG GCA TTT GGC GGC TTC ATT CTG TGT AAG GAA GGA GAA 704 Glu Ser Gln ValAla Phe Gly Gly Phe Ile Leu Cys Lys Glu Gly Glu            165                 170                 175 GAT GAA CAC CCACAA TGC CTG AAC TCC CAG CCC CAT GCC CGT GGG TCG 752 Asp Glu His Pro GlnCys Leu Asn Ser Gln Pro His Ala Arg Gly Ser        180                 185                 190 TCC CGC GCC ATC TTCTCC GTG GGC CCC GTG AGC CCG AAT CGC AGG TGG 800 Ser Arg Ala Ile Phe SerVal Gly Pro Val Ser Pro Asn Arg Arg Trp    195                 200                 205 TCG CAC AGG TGC TAT GGTTAT GAC TTG AAC TCT CCC TAT GTG TGG TCT 848 Ser His Arg Cys Tyr Gly TyrAsp Leu Asn Ser Pro Tyr Val Trp Ser210                 215                 220                 225 TCA CCCAGT GAT CTC CTG GAG CTC CTG GTC CCA GGT GTT TCT AAG AAG 896 Ser Pro SerAsp Leu Leu Glu Leu Leu Val Pro Gly Val Ser Lys Lys                230                 235                 240 CCA TCA CTCTCA GTG CAG CCG GGT CCT GTC GTG GCC CCT GGG GAA AGC 944 Pro Ser Leu SerVal Gln Pro Gly Pro Val Val Ala Pro Gly Glu Ser            245                 250                 255 CTG ACC CTC CAGTGT GTC TCT GAT GTC GGC TAT GAC AGA TTT GTT CTG 992 Leu Thr Leu Gln CysVal Ser Asp Val Gly Tyr Asp Arg Phe Val Leu        260                 265                 270 TAC AAG GAG GGG GAACGT GAC CTT CGC CAG CTC CCT GGC CGG CAG CCC 1040 Tyr Lys Glu Gly Glu ArgAsp Leu Arg Gln Leu Pro Gly Arg Gln Pro    275                 280                 285 CAG GCT GGG CTC TCC CAGGCC AAC TTC ACC CTG GGC CCT GTG AGC CGC 1088 Gln Ala Gly Leu Ser Gln AlaAsn Phe Thr Leu Gly Pro Val Ser Arg290                 295                 300                 305 TCC TACGGG GGC CAG TAC AGA TGC TAC GGT GCA TAC AAC CTC TCC TCC 1136 Ser Tyr GlyGly Gln Tyr Arg Cys Tyr Gly Ala Tyr Asn Leu Ser Ser                310                 315                 320 GAG TGG TCGGCC CCC AGC GAC CCC CTG GAC ATC CTG ATC ACA GGA CAG 1184 Glu Trp Ser AlaPro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln            325                 330                 335 ATC CAT GGC ACACCC TTC ATC TCA GTG CAG CCA GGC CCC ACA GTG GCC 1232 Ile His Gly Thr ProPhe Ile Ser Val Gln Pro Gly Pro Thr Val Ala        340                 345                 350 TCA GGA GAG AAC GTGACC CTG CTG TGT CAG TCA TGG CGG CAG TTC CAC 1280 Ser Gly Glu Asn Val ThrLeu Leu Cys Gln Ser Trp Arg Gln Phe His    355                 360                 365 ACT TTC GTT CTG ACC AAGGCG GGA GCA GCT GAT GCC CCA CTC CGT CTA 1328 Thr Phe Leu Leu Thr Lys AlaGly Ala Ala Asp Ala Pro Leu Arg Leu370                 375                 380                 385 AGA TCAATA CAC GAA TAT CCT AAG TAC CAG GCT GAA TTC CCC ATG AGT 1376 Arg Ser IleHis Glu Tyr Pro Lys Tyr Gln Ala Glu Phe Pro Met Ser                390                 395                 400 CCT GTG ACCTCA GCC CAC GCG GGG ACC TAC AGG ACC CTC CAT GGG TTC 1424 Pro Val Thr SerAla His Ala Gly Thr Tyr Arg Thr Leu His Gly Phe            405                 410                 415 CAG CCC CCC ACCCAC CGG TCC CAT CTC CAC ACC TGC AGG CCC 1466 Gln Pro Pro Thr His Arg SerHis Leu His Thr Cys Arg Pro        420                 425                 430 TGAGGACCAGCCCCTCACCC CCACTGGGTC GGATCCCCAA AGTGGTCTGG GAAGGCACCT 1526 GGGGGTTGTGATCGGCATCT TGGTGGCCGT CGTCCTACTG CTCCTCCTCC TCCTCCTCCT 1586 CTTCCTCATCCTCCGACATC GACGTCAGGG CAAACACTGG ACATCGACCC AGAGAAAGGC 1646 TGATTTCCAACATCCTGCAG GGGCTGTGGG GCCAGAGCCC ACAGACAGAG GCCTGCAGTG 1706 GAGGTCCAGCCCAGCTGCCG ACGCCCAGGA AGAAAACCTC TATGCTGCCG TGAAGGACAC 1766 ACAGCCTGAAGATGGGGTGG AGATGGACAC TCGGGCTGCT GCATCTGAAG CCCCCCAGGA 1826 TGTGACCTACGCCCAGCTGC ACAGCTTGAC CCTCAGACGG AAGGCAACTG AGCCTCCTCC 1886 ATCCCAGGAAAGGGAACCTC CAGCTGAGCC CAGCATCTAC GCCACCCTGG CCATCCACTA 1946 GCCCGGAGGGTACGCAGACT CCACACTCAG TAGAAGGAGA CTCAGGACTG CTGAAGGCAC 2006 GGGAGCTGCCCCCAGTGGAC ACCAATGAAC CCCAGTCAGC CTGGACCCCT AACAAAGACC 2066 ATGAGGAGATGCTGGGAACT TTGGGACTCA CTTGATTCTG CAGTCGAAAT AACTAATATC 2126 CCTACATTTTTTAATTAAAG CAACAGACTT CTCAATAATC AATGAGTTAA CCGAGAAAAC 2186 TAAAAAAAAAAAAA 2200 A KTE03 embodiment designated KLM67 (SEQ ID NO: 21 and 22):GCCACACGCA GCTCAGCCTG GGCGGCACAG CCAGATGCGA GATGCGTCTC TGCTGATCTG 60AGTCTGCCTG CAGCATGGAC CTGGGTCTTC CCTGAAGCAT CTCCAGGGCT GGAGGGACGA 120CTGCCATGCA CCGAGGGCTC ATCCATCCAC AGAGCAGGGC AGTGGGAGGA GACGGC 176 ATGACC CCC ATC CTC ACG GTC CTG ATC TGT CTC GGG CTG AGT CTG GGC 224 Met ThrPro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly  1               5                  10                  15 CCC CGG ACCCAC GTG CAG GCA GGG CAC CTC CCC AAG CCC ACC CTC TGG 272 Pro Arg Thr HisVal Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp             20                  25                  30 GCT GAA CCA GGCTCT GTG ATC ACC CAG GGG AGT CCT GTG ACC CTC AGG 320 Ala Glu Pro Gly SerVal Ile Thr Gln Gly Ser Pro Val Thr Leu Arg         35                  40                  45 TGT CAG GGG GGC CAGGAG ACC CAG GAG TAC CGT CTA TAT AGA GAA AAG 368 Cys Gln Gly Gly Gln GluThr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys     50                  55                  60 AAA ACA GCA CCC TGG ATTACA CGG ATC CCA CAG GAG CTT GTG AAG AAG 416 Lys Thr Ala Pro Trp Ile ThrArg Ile Pro Gln Glu Leu Val Lys Lys 65                  70                  75                  80 GGC CAGTTC CCC ATC CCA TCC ATC ACC TGG GAA CAT GCA GGG CGG TAT 464 Gly Gln PhePro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr                 85                  90                  95 CGC TGT TACTAT GGT AGC GAC ACT GCA GGC CGC TCA GAG AGC AGT GAC 512 Arg Cys Tyr TyrGly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp            100                 105                 110 CCC CTG GAG CTGGTG GTG ACA GGA GCC TAC ATC AAA CCC ACC CTC TCA 560 Pro Leu Glu Leu ValVal Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser        115                 120                 125 GCC CAG CCC AGC CCCGTG GTG AAC TCA GGA GGG AAT GTA ACC CTC CAG 608 Ala Gln Pro Ser Pro ValVal Asn Ser Gly Gly Asn Val Thr Leu Gln    130                 135                 140 TGT GAC TCA CAG GTG GCATTT GAT GGC TTC ATT CTG TGT AAG GAA GGA 656 Cys Asp Ser Gln Val Ala PheAsp Gly Phe Ile Leu Cys Lys Glu Gly145                 150                 155                 160 GAA GATGAA CAC CCA CAA TGC CTG AAC TCC CAG CCC CAT GCC CGT GGG 704 Glu Asp GluHis Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly                165                 170                 175 TCG TCC CGCGCC ATC TTC TCC GTG GGC CCC GTG AGC CCG AGT CGC AGG 752 Ser Ser Arg AlaIle Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg            180                 185                 190 TGG TGG TAC AGGTGC TAT GCT TAT GAC TCG AAC TCT CCC TAT GAG TGG 800 Trp Trp Tyr Arg CysTyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp        195                 200                 205 TCT CTA CCC AGT GATCTC CTG GAG CTC CTG GTC CTA GGT GTT TCT AAG 848 Ser Leu Pro Ser Asp LeuLeu Glu Leu Leu Val Leu Gly Val Ser Lys    210                 215                 220 AAC CCA TCA CTC TCA GTGCAG CCA GGT CCT ATC GTG GCC CCT GAG GAG 896 Lys Pro Ser Leu Ser Val GlnPro Gly Pro Ile Val Ala Pro Glu Glu225                 230                 235                 240 ACC CTGACT CTG CAG TGT GGC TCT GAT GCT GGC TAC AAC AGA TTT GTT 944 Thr Leu ThrLeu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val                245                 250                 255 CTG TAT AAGGAC GGG GAA CGT GAC TTC CTT CAG CTC GCT GGC GCA CAG 992 Leu Tyr Lys AspGly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln            260                 265                 270 CCC CAG GCT GGGCTC TCC CAG GCC AAC TTC ACC CTG GGC CCT GTG AGC 1040 Pro Gln Ala Gly LeuSer Gln Ala Asn Phe Thr Leu Gly Pro Val Ser        275                 280                 285 CGC TCC TAC GGG GGCCAG TAC AGA TGC TAC GGT GCA CAC AAC CTC TCC 1088 Arg Ser Tyr Gly Gly GlnTyr Arg Cys Tyr Gly Ala His Asn Leu Ser    290                 295                 300 TCC GAG TGG TCG GCC CCCAGC GAC CCC CTG GAC ATC CTG ATC GCA GGA 1136 Ser Glu Trp Ser Ala Pro SerAsp Pro Leu Asp Ile Leu Ile Ala Gly305                 310                 315                 320 CAG TTCTAT GAC AGA GTC TCC CTC TCG GTG CAG CCG GGC CCC ACG GTG 1184 Gln Phe TyrAsp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val                325                 330                 335 GCC TCA GGAGAG AAC GTG ACC CTG CTG TGT CAG TCA CAG GGA TGG ATG 1232 Ala Ser Gly GluAsn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met            340                 345                 350 CAA ACT TTC CTTCTG ACC AAG GAG GGG GCA GCT GAT GAC CCA TGG GGT 1280 Gln Thr Phe Leu LeuThr Lys Glu Gly Ala Ala Asp Asp Pro Trp Arg        355                 360                 365 CTA AGA TCA ACG TACCAA TCT CAA AAA TAC CAG GCT GAA TTC CCC ATG 1328 Leu Arg Ser Thr Tyr GlnSer Gln Lys Tyr Gln Ala Glu Phe Pro Met    370                 375                 380 GGT CCT GTG ACC TCA GCCCAT GCG GGG ACC TAC AGG TGC TAC GGC TCA 1376 Gly Pro Val Thr Ser Ala HisAla Gly Thr Tyr Arg Cys Tyr Gly Ser385                 390                 395                 400 CAG AGCTCC AAA CCC TAC CTG CTG ACT CAC CCC AGT GAC CCC CTG GAG 1424 Gln Ser SerLys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu                405                 410                 415 CTC GTG GTCTCA GGA CCG TCT GGG GGC CCC AGC TCC CCG ACA ACA GGC 1472 Leu Val Val SerGly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly            420                 425                 430 CCC ACC TCC ACATCT GGC CCT GAG GAC CAG CCC CTC ACC CCC ACC GGG 1520 Pro Thr Ser Thr SerGly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly        435                 440                 445 TCG GAT CCC CAG AGTGGT CTG GGA AGG CAC CTG GGG GTT GTG ATC GGC 1568 Ser Asp Pro Gln Ser GlyLeu Gly Arg His Leu Gly Val Val Ile Gly    450                 455                 460 ATC TTG GTG GCC GTC ATCCTA CTG CTC CTC CTC CTC CTC CTG CTG TTC 1616 Ile Leu Val Ala Val Ile LeuLeu Leu Leu Leu Leu Leu Leu Leu Phe465                 470                 475                 480 CTC ATCCTC CGA CAT CGA CGT CAG GGC AAA CAC TGG ACA TCG ACC CAG 1664 Leu Ile LeuArg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln                485                 490                 495 AGA AAG GCTGAT TTC CAA CAT CCT GCA GGG GCT GTG GGG CCA GAG CCC 1712 Arg Lys Ala AspPhe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro            500                 505                 510 ACA GAC AGA CGCCTG CAG TGG AGG TCC AGC CCA GCT GCC GAT GCC CAG 1760 Thr Asp Arg Arg LeuGln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln        515                 520                 525 GAA GAA AAC CTC TATGCT GCC GTG AAG CAC ACA CAG CCT GAG GAT GGG 1808 Glu Glu Asn Leu Tyr AlaAla Val Lys His Thr Gln Pro Glu Asp Gly    530                 535                 540 GTG GAG ATG GAC ACT CGGCAG AGC CCA CAC GAT GAA GAC CCC CAG GCA 1856 Val Glu Met Asp Thr Arg GlnSer Pro His Asp Glu Asp Pro Gln Ala545                 550                 555                 560 GTG ACGTAT GCC GAG GTG AAA CAC TCC AGA CCT AGG AGA GAA ATG GCT 1904 Val Thr TyrAla Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala                565                 570                 575 TCT CCT CCTTCC CCA CTG TCT GGG GAA TTC CTG GAC ACA AAG GAC AGA 1952 Ser Pro Pro SerPro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg            580                 585                 590 CAG GCG GAA GAGGAC AGG CAG ATG GAC ACT GAG GCT GCT GCA TCT GAA 2000 Gln Ala Glu Glu AspArg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu        595                 600                 605 GCC CCC CAG GAT GTGACC TAC GCC CAG CTG CAC AGC TTG ACC CTT AGA 2048 Ala Pro Gln Asp Val ThrTyr Ala Gln Leu His Ser Leu Thr Leu Arg    610                 615                 620 CGG AAG GCA ACT GAG CCTCCT CCA TCC CAG GAA GGG CCC TCT CCA GCT 2096 Arg Lys Ala Thr Glu Pro ProPro Ser Gln Glu Gly Pro Ser Pro Ala625                 630                 635                 640 GTG CCCAGC ATC TAC GCC ACT CTG GCC ATC CAC TAG CCCAGGGGGG 2142 Val Pro Ser IleTyr Ala Thr Leu Ala Ile His  *                 645                 650GACGCAGACC CCACACTCCA TGGAGTCTGG AATGCATGGG AGCTGCCCCC CCAGTGGACA 2202CCATTGGACC CCACCCAGCC TGGATCTACC CCAGGAGACT CTGGGAACTT TTAGGGGTCA 2262CTCAATTCTG CAGTATAAAT AACTAATGTC TCTACAATTT TGAAATAAAG CAACAGACTT 2322CTCAATAATC AATGAAGTAG CTGAGAAAAC TAAGTCAGAA AGTGCATTAA ACTGAATCAC 2382AATGTAAATA TTACACATCA AGCGATGAAA CTGGAAAACT ACAAGCCACG AATGAATGAA 2442TTAGGAAAGA AAAAAAGTAG GAAATGAATG ATCTTGGCTT TCCTATAAGA AATTTAGGGC 2502AGGGCACGGT GGCTCACGCC TGTAATTCCA GCACTTTGGG AGGCCGAGGC GGGCAGATCA 2562CGAGTTCAGG AGATCGAGAC CATCTTGGCC AACATGGTGA AACCCTGTCT CTCCTAAAAA 2622TACAAAAATT AGCTGGATGT GGTGGCAGTG CCTGTAATCC CAGCTATTTG GGAGGCTGAG 2682GCAGGAGAAT CGCTTGAACC AGGGAGTCAG AGGTTTCAGT GAGCCAAGAT CGCACCACTG 2742CTCTCCAGCC TGGCGACAGA GGGAGACTCC ATCTCAAATT AAAAAAAA 2790

[0056] The peptide segments can also be used to produce appropriateoligonucleotides to screen a library to determine the presence of asimilar gene, e.g., an identical or polymorphic variant, or to identifya monocyte. The genetic code can be used to select appropriateoligonucleotides useful as probes for screening. In combination withpolymerase chain reaction (PCR) techniques, synthetic oligonucleotideswill be useful in selecting desired clones from a library.

[0057] Complementary sequences will also be used as probes or primers.Based upon identification of the likely amino terminus, other peptidesshould be particularly useful, e.g., coupled with anchored vector orpoly-A complementary PCR techniques or with complementary DNA of otherpeptides.

[0058] Techniques for nucleic acid manipulation of genes encoding thesemonocyte proteins, e.g., subcloning nucleic acid sequences encodingpolypeptides into expression vectors, labeling probes, DNAhybridization, and the like are described generally in Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY, which isincorporated herein by reference and hereinafter referred to as“Sambrook, et al.” See also, Coligan, et al. (1987 and periodicsupplements) Current Protocols in Molecular Biology Greene/Wiley, NewYork, N.Y., referred to as “Coligan, et al.”

[0059] There are various methods of isolating the DNA sequences encodingthese monocyte proteins. For example, DNA is isolated from a genomic orcDNA library using labeled oligonucleotide probes having sequencesidentical or complementary to the sequences disclosed herein.Full-length probes may be used, or oligonucleotide probes may begenerated by comparison of the sequences disclosed with other proteinsand selecting specific primers. Such probes can be used directly inhybridization assays to isolate DNA encoding monocyte proteins, orprobes can be designed for use in amplification techniques such as PCR,for the isolation of DNA encoding monocyte proteins.

[0060] To prepare a cDNA library, mRNA is isolated from cells whichexpress the monocyte protein. cDNA is prepared from the mRNA and ligatedinto a recombinant vector. The vector is transfected into a recombinanthost for propagation, screening and cloning. Methods for making andscreening cDNA libraries are well known. See Gubler and Hoffman (1983)Gene 25:263-269; Sambrook, et al.; or Coligan, et al.

[0061] For a genomic library, the DNA can be extracted from tissue andeither mechanically sheared or enzymatically digested to yield fragmentsof about 12-20 kb. The fragments are then separated by gradientcentrifugation and cloned in bacteriophage lambda vectors. These vectorsand phage are packaged in vitro, as described, e.g., in Sambrook, et al.or Coligan, et al. Recombinant phage are analyzed by plaquehybridization as described in Benton and Davis (1977) Science196:180-182. Colony hybridization is carried out as generally describedin, e.g., Grunstein, et al. (1975) Proc. Natl. Acad. Sci. USA72:3961-3965.

[0062] DNA encoding a monocyte protein can be identified in either cDNAor genomic libraries by its ability to hybridize with the nucleic acidprobes described herein, for example in colony or plaque hybridizationexperiments. The corresponding DNA regions are isolated by standardmethods familiar to those of skill in the art. See Sambrook, et al.

[0063] Various methods of amplifying target sequences, such as thepolymerase chain reaction, can also be used to prepare DNA encodingmonocyte proteins. Polymerase chain reaction (PCR) technology is used toamplify such nucleic acid sequences directly from mRNA, from cDNA, andfrom genomic libraries or cDNA libraries. The isolated sequencesencoding monocyte proteins may also be used as templates for PCRamplification.

[0064] In PCR techniques, oligonucleotide primers complementary to two5′ regions in the DNA region to be amplified are synthesized. Thepolymerase chain reaction is then carried out using the two primers. SeeInnis, et al. (eds.) (1990) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif. Primers can be selectedto amplify the entire regions encoding a selected full-length monocyteprotein or to amplify smaller DNA segments as desired. Once such regionsare PCR-amplified, they can be sequenced and oligonucleotide probes canbe prepared from sequence obtained using standard techniques. Theseprobes can then be used to isolate DNAs encoding other forms of themonocyte proteins.

[0065] Oligonucleotides for use as probes are chemically synthesizedaccording to the solid phase phosphoramidite triester method firstdescribed by Beaucage and Carruthers (1983) Tetrahedron Lett.22(20):1859-1862, or using an automated synthesizer, as described inNeedham-VanDevanter, et al. (1984) Nucleic Acids Res. 12:6159-6168.Purification of oligonucleotides is performed e.g., by native acrylamidegel electrophoresis or by anion-exchange HPLC as described in Pearsonand Regnier (1983) J. Chrom. 255:137-149. The sequence of the syntheticoligonucleotide can be verified using the chemical degradation method ofMaxam and Gilbert in Grossman and Moldave (eds.) (1980) Methods inEnzymology 65:499-560 Academic Press, New York.

[0066] An isolated nucleic acid encoding a human protein which is a typeI transmembrane protein comprising an extracellular portioncharacterized by Ig-like domains, indicating that this gene encodes areceptor member of the Ig superfamily. This clone has been designatedFDF03. Its nucleotide sequence and corresponding open reading frame areprovided in SEQ ID NO: 1 and 2, respectively. An N-terminal hydrophobicsequence, e.g., a putative signal sequence, corresponds to about aminoacid residues −19(met) to −1(leu), and a internal hydrophobic segment,corresponding to a putative transmembrane segment runs from aroundala177 to leu199. Other mammalian counterparts should become available,e.g., a partial rodent gene is described in SEQ ID NO: 3 and 4. Standardtechniques will allow isolation of other counterparts, or to extendpartial sequences.

[0067] A second human monocyte cell clone was isolated, designated YE01,is related to the receptors for Fc gamma and/or Fc alpha. This has alsobeen referred to as DNAX Leukocyte Associated Immunoglobulin-likeReceptor (DLAIR). See also Meyaard, et al. (1997) Immunity 7:283-290,which was published by the inventors after the priority date of thisapplication, and is incorporated herein by reference. This protein isreferred to herein as an Fc gamma/alpha receptor and is described in SEQID NO: 5 and 6. Another human isolate is described in SEQ ID NO: 7 and8. A soluble form of the receptor is encoded in SEQ ID NO: 9 and 10.While the gene was initially described as a monocyte derived gene,expression analysis indicates that it is more specific for expression onlymphocytes. Thus, in the case of YE01, the descriptor “monocyte gene”may indicate its original identification in a population enriched forthat cell type, though it may have also contained some other cell types.Sequence analysis suggests YE01 is a member of the Ig superfamily ofreceptors, and is closely related to the CD8 family, which contain aV1J-type fold, particularly the Fc receptors alpha and/or gamma. Becauseit contains an ITAM-like motif, the protein may well be a lymphocyteversion of the Killer Inhibitory Receptors (KIR), which send a negativesignal to inhibit killer cell function. This protein exhibits similarfunction in inhibiting lymphocyte effector function, e.g., antigenpresentation or subsequent response initiation.

[0068] In particular, signaling through the molecule recognized by DX26mAb (designated DNAX Leukocyte Associated Immunoglobulin-like Receptor(DLAIR)), delivers a negative signal to NK cell clones that preventstheir killing specific target cells. However, the molecule is expressedon other lymphocytes, including T cells and monocytes. Thus, the DX26antibody probably represents an antibody which both inhibits NK andcytotoxic T cell killing, and the monocyte distribution suggests thatthe molecule may inhibit monocyte-mediated or lymphocyte-mediatedeffector functions.

[0069] A third monocyte gene was isolated and designated KTE03, and isrepresented by six related embodiments, designated YYB01, YYB04 (forms 1and 2), (KIR-Like Molecule) KLM63, KLM66, and KLM67. See SEQ ID NO:11-22. Note that a possible splice variant, which may encode a variantprotein form, has been detected.

[0070] This invention provides isolated DNA or fragments to encode amonocyte protein, as described. In addition, this invention providesisolated or recombinant DNA which encodes a biologically active proteinor polypeptide which is capable of hybridizing under appropriateconditions, e.g., high stringency, with the DNA sequences describedherein. Said biologically active protein or polypeptide can be anaturally occurring form, or a recombinant protein or fragment, and havean amino acid sequence as disclosed in SEQ ID NO: 2 or 4; 6, 8, or 10;or 12, 14, 16, 18, 20, or 22. Preferred embodiments will be full lengthnatural isolates, e.g., from a primate. In glycosylated form, theproteins should exhibit larger sizes. Further, this inventionencompasses the use of isolated or recombinant DNA, or fragmentsthereof, which encode proteins which are homologous to each respectivemonocyte protein. The isolated DNA can have the respective regulatorysequences in the 5′ and 3′ flanks, e.g., promoters, enhancers, poly-Aaddition signals, and others.

[0071] IV. Making Monocyte Gene Products

[0072] DNAs which encode these monocyte proteins or fragments thereofcan be obtained by chemical synthesis, screening cDNA libraries, or byscreening genomic libraries prepared from a wide variety of cell linesor tissue samples.

[0073] These DNAs can be expressed in a wide variety of host cells forthe synthesis of a full-length protein or fragments which can, e.g., beused to generate polyclonal or monoclonal antibodies; for bindingstudies; for construction and expression of modified molecules; and forstructure/function studies. Each of these monocyte proteins or theirfragments can be expressed in host cells that are transformed ortransfected with appropriate expression vectors. These molecules can besubstantially purified to be free of protein or cellular contaminants,other than those derived from the recombinant host, and therefore areparticularly useful in pharmaceutical compositions when combined with apharmaceutically acceptable carrier and/or diluent. The antigen, orportions thereof, may be expressed as fusions with other proteins.

[0074] Expression vectors are typically self-replicating DNA or RNAconstructs containing the desired monocyte gene or its fragments,usually operably linked to suitable genetic control elements that arerecognized in a suitable host cell. These control elements are capableof effecting expression within a suitable host. The specific type ofcontrol elements necessary to effect expression will depend upon theeventual host cell used. Generally, the genetic control elements caninclude a prokaryotic promoter system or a eukaryotic promoterexpression control system, and typically include a transcriptionalpromoter, an optional operator to control the onset of transcription,transcription enhancers to elevate the level of mRNA expression, asequence that encodes a suitable ribosome binding site, and sequencesthat terminate transcription and translation. Expression vectors alsousually contain an origin of replication that allows the vector toreplicate independently from the host cell.

[0075] The vectors of this invention contain DNAs which encode thevarious monocyte proteins, or a fragment thereof, typically encoding,e.g., a biologically active polypeptide, or protein. The DNA can beunder the control of a viral promoter and can encode a selection marker.This invention further contemplates use of such expression vectors whichare capable of expressing eukaryotic cDNA coding for a monocyte proteinin a prokaryotic or eukaryotic host, where the vector is compatible withthe host and where the eukaryotic cDNA coding for the protein isinserted into the vector such that growth of the host containing thevector expresses the cDNA in question. Usually, expression vectors aredesigned for stable replication in their host cells or for amplificationto greatly increase the total number of copies of the desirable gene percell. It is not always necessary to require that an expression vectorreplicate in a host cell, e.g., it is possible to effect transientexpression of the protein or its fragments in various hosts usingvectors that do not contain a replication origin that is recognized bythe host cell. It is also possible to use vectors that cause integrationof a monocyte gene or its fragments into the host DNA by recombination,or to integrate a promoter which controls expression of an endogenousgene.

[0076] Vectors, as used herein, comprise plasmids, viruses,bacteriophage, integratable DNA fragments, and other vehicles whichenable the integration of DNA fragments into the genome of the host.Expression vectors are specialized vectors which contain genetic controlelements that effect expression of operably linked genes. Plasmids arethe most commonly used form of vector but all other forms of vectorswhich serve an equivalent function are suitable for use herein. See,e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors: ALaboratory Manual Elsevier, N.Y.; and Rodriquez, et al. (eds.) (1988)Vectors: A Survey of Molecular Cloning Vectors and Their UsesButtersworth, Boston, Mass.

[0077] Suitable host cells include prokaryotes, lower eukaryotes, andhigher eukaryotes. Prokaryotes include both gram negative and grampositive organisms, e.g., E. coli and B. subtilis. Lower eukaryotesinclude yeasts, e.g., S. cerevisiae and Pichia, and species of the genusDictyostelium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

[0078] Prokaryotic host-vector systems include a wide variety of vectorsfor many different species. As used herein, E. coli and its vectors willbe used generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 or itsderivatives. Vectors that can be used to express monocyte proteins orfragments include, but are not limited to, such vectors as thosecontaining the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipppromoter (the pIN-series); lambda-pP or pR promoters (pOTS); or hybridpromoters such as ptac (pDR540). See Brosius, et al. (1988) “ExpressionVectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters”, inRodriguez and Denhardt (eds.) Vectors: A Survey of Molecular CloningVectors and Their Uses 10:205-236 Buttersworth, Boston, Mass.

[0079] Lower eukaryotes, e.g., yeasts and Dictyostelium, may betransformed with monocyte gene sequence containing vectors. For purposesof this invention, the most common lower eukaryotic host is the baker'syeast, Saccharomyces cerevisiae. It will be used generically torepresent lower eukaryotes although a number of other strains andspecies are also available. Yeast vectors typically consist of areplication origin (unless of the integrating type), a selection gene, apromoter, DNA encoding the desired protein or its fragments, andsequences for translation termination, polyadenylation, andtranscription termination. Suitable expression vectors for yeast includesuch constitutive promoters as 3-phosphoglycerate kinase and variousother glycolytic enzyme gene promoters or such inducible promoters asthe alcohol dehydrogenase 2 promoter or metallothionine promoter.Suitable vectors include derivatives of the following types:self-replicating low copy number (such as the YRp-series),self-replicating high copy number (such as the YEp-series); integratingtypes (such as the YIp-series), or mini-chromosomes (such as theYCp-series).

[0080] Higher eukaryotic tissue culture cells are the preferred hostcells for expression of the monocyte protein. In principle, most anyhigher eukaryotic tissue culture cell line may be used, e.g., insectbaculovirus expression systems, whether from an invertebrate orvertebrate source. However, mammalian cells are preferred to achieveproper processing, both cotranslationally and posttranslationally.Transformation or transfection and propagation of such cells is routine.Useful cell lines include HeLa cells, Chinese hamster ovary (CHO) celllines, baby rat kidney (BRK) cell lines, insect cell lines, bird celllines, and monkey (COS) cell lines. Expression vectors for such celllines usually include an origin of replication, a promoter, atranslation initiation site, RNA splice sites (e.g., if genomic DNA isused), a polyadenylation site, and a transcription termination site.These vectors also may contain a selection gene or amplification gene.Suitable expression vectors may be plasmids, viruses, or retrovirusescarrying promoters derived, e.g., from such sources as from adenovirus,SV40, parvoviruses, vaccinia virus, or cytomegalovirus. Representativeexamples of suitable expression vectors include pcDNA1; pCD, seeOkayama, et al. (1985) Mol. Cell Biol. 5:1136-1142; pMC1eo Poly-A, seeThomas, et al. (1987) Cell 51:503-512; and a baculovirus vector such aspAC 373 or pAC 610.

[0081] In certain instances, the monocyte proteins need not beglycosylated to elicit biological responses in certain assays. However,it will often be desirable to express a monocyte polypeptide in a systemwhich provides a specific or defined glycosylation pattern. In thiscase, the usual pattern will be that provided naturally by theexpression system. However, the pattern will be modifiable by exposingthe polypeptide, e.g., in unglycosylated form, to appropriateglycosylating proteins introduced into a heterologous expression system.For example, a monocyte gene may be co-transformed with one or moregenes encoding mammalian or other glycosylating enzymes. It is furtherunderstood that over glycosylation may be detrimental to monocyteprotein biological activity, and that one of skill may perform routinetesting to optimize the degree of glycosylation which confers optimalbiological activity.

[0082] A monocyte protein, or a fragment thereof, may be engineered tobe phosphatidyl inositol (PI) linked to a cell membrane, but can beremoved from membranes by treatment with a phosphatidyl inositolcleaving enzyme, e.g., phosphatidyl inositol phospholipase-C. Thisreleases the antigen in a biologically active form, and allowspurification by standard procedures of protein chemistry. See, e.g., Low(1989) Biochem. Biophys. Acta 988:427-454; Tse, et al. (1985) Science230:1003-1008; Brunner, et al. (1991) J. Cell Biol. 114:1275-1283; andColigan, et al. (eds.) (1996 and periodic supplements) Current Protocolsin Protein Science, John Wiley & Sons, New York, N.Y.

[0083] Now that these monocyte proteins have been characterized,fragments or derivatives thereof can be prepared by conventionalprocesses for synthesizing peptides. These include processes such as aredescribed in Stewart and Young (1984) Solid Phase Peptide SynthesisPierce Chemical Co., Rockford, Ill.; Bodanszky and Bodanszky (1984) ThePractice of Peptide Synthesis Springer-Verlag, New York, N.Y.; andBodanszky (1984) The Principles of Peptide Synthesis Springer-Verlag,New York, N.Y. See also Merrifield (1986) Science 232:341-347; andDawson, et al. (1994) Science 266:776-779. For example, an azideprocess, an acid chloride process, an acid anhydride process, a mixedanhydride process, an active ester process (for example, p-nitrophenylester, N-hydroxysuccinimide ester, or cyanomethyl ester), acarbodiimidazole process, an oxidative-reductive process, or adicyclohexylcarbodiimide (DCCD)/additive process can be used. Solidphase and solution phase syntheses are both applicable to the foregoingprocesses.

[0084] The prepared protein and fragments thereof can be isolated andpurified from the reaction mixture by means of peptide separation, forexample, by extraction, precipitation, electrophoresis and various formsof chromatography, and the like. The monocyte proteins of this inventioncan be obtained in varying degrees of purity depending upon the desireduse. Purification can be accomplished by use of known proteinpurification techniques or by the use of the antibodies or bindingpartners herein described, e.g., in immunoabsorbant affinitychromatography. This immunoabsorbant affinity chromatography is carriedout by first linking the antibodies to a solid support and contactingthe linked antibodies with solubilized lysates of appropriate sourcecells, lysates of other cells expressing the protein, or lysates orsupernatants of cells producing the proteins as a result of DNAtechniques, see below.

[0085] Multiple cell lines may be screened for one which expresses saidprotein at a high level compared with other cells. Various cell lines,e.g., a mouse thymic stromal cell line TA4, is screened and selected forits favorable handling properties. Natural monocyte cell proteins can beisolated from natural sources, or by expression from a transformed cellusing an appropriate expression vector. Purification of the expressedprotein is achieved by standard procedures, or may be combined withengineered means for effective purification at high efficiency from celllysates or supernatants. FLAG or His₆ segments can be used for suchpurification features.

[0086] V. Antibodies

[0087] Antibodies can be raised to these various monocyte proteins,including individual, polymorphic, allelic, strain, or species variants,and fragments thereof, both in their naturally occurring (full-length)forms and in their recombinant forms. Additionally, antibodies can beraised to monocyte proteins in either their active forms or in theirinactive forms. Anti-idiotypic antibodies may also be used.

[0088] a. Antibody Production

[0089] A number of immunogens may be used to produce antibodiesspecifically reactive with these monocyte proteins. Recombinant proteinis the preferred immunogen for the production of monoclonal orpolyclonal antibodies. Naturally occurring protein may also be usedeither in pure or impure form. Synthetic peptides made using the humanmonocyte protein sequences described herein may also used as animmunogen for the production of antibodies to the monocyte protein.Recombinant protein can be expressed in eukaryotic or prokaryotic cellsas described herein, and purified as described. The product is theninjected into an animal capable of producing antibodies. Eithermonoclonal or polyclonal antibodies may be generated for subsequent usein immunoassays to measure the protein.

[0090] Methods of producing polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen, preferably a purified protein,is mixed with an adjuvant and animals are immunized with the mixture.The animals immune response to the immunogen preparation is monitored bytaking test bleeds and determining the titer of reactivity to themonocyte protein of interest. When appropriately high titers of antibodyto the immunogen are obtained, blood is collected from the animal andantisera are prepared. Further fractionation of the antisera to enrichfor antibodies reactive to the protein can be done if desired. See,e.g., Harlow and Lane.

[0091] Monoclonal antibodies may be obtained by various techniquesfamiliar to those skilled in the art. Briefly, spleen cells from ananimal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell. See, e.g., Kohler and Milstein (1976) Eur.J. Immunol. 6:511-519, which is incorporated herein by reference.Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods knownin the art. Colonies arising from single immortalized cells are screenedfor production of antibodies of the desired specificity and affinity forthe antigen, and yield of the monoclonal antibodies produced by suchcells may be enhanced by various techniques, including injection intothe peritoneal cavity of a vertebrate host. Alternatively, one mayisolate DNA sequences which encode a monoclonal antibody or a bindingfragment thereof by screening a DNA library from human B cells accordingto the general protocol outlined by Huse, et al. (1989) Science246:1275-1281.

[0092] Antibodies, including binding fragments and single chainversions, against predetermined fragments of these monocyte proteins canbe raised by immunization of animals with conjugates of the fragmentswith carrier proteins as described above. Monoclonal antibodies areprepared from cells secreting the desired antibody. These antibodies canbe screened for binding to normal or defective monocyte proteins, orscreened for agonistic or antagonistic activity. These monoclonalantibodies will usually bind with at least a K_(D) of about 1 mM, moreusually at least about 300 μM, typically at least about 100 μM, moretypically at least about 30 μM, preferably at least about 10 μM, andmore preferably at least about 3 μM or better. Standard methods areavailable for selection of high affinity and selective antibodypreparations.

[0093] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basicand Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane (1988) Antibodies:A Laboratory Manual CSH Press; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andparticularly in Kohler and Milstein (1975) Nature 256:495-497, whichdiscusses one method of generating monoclonal antibodies. Summarizedbriefly, this method involves injecting an animal with an immunogen toinitiate a humoral immune response. The animal is then sacrificed andcells taken from its spleen, which are then fused with myeloma cells.The result is a hybrid cell or “hybridoma” that is capable ofreproducing in vitro. The population of hybridomas is then screened toisolate individual clones, each of which secretes a single antibodyspecies to the immunogen. In this manner, the individual antibodyspecies obtained are the products of immortalized and cloned single Bcells from the immune animal generated in response to a specific siterecognized on the immunogenic substance.

[0094] Other suitable techniques involve selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced. See, Cabilly, U.S. Pat. No. 4,816,567; and Queen, etal. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033.

[0095] The antibodies of this invention can also be used for affinitychromatography in isolating each monocyte protein. Columns can beprepared where the antibodies are linked to a solid support, e.g.,particles, such as agarose, SEPHADEX, or the like, where a cell lysatemay be passed through the column, the column washed, followed byincreasing concentrations of a mild denaturant, whereby purifiedmonocyte protein will be released.

[0096] The antibodies may also be used to screen expression librariesfor particular expression products. Usually the antibodies used in sucha procedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

[0097] Antibodies to monocyte proteins may be used for the analysis or,or identification of specific cell population components which expressthe respective protein. By assaying the expression products of cellsexpressing monocyte proteins it is possible to diagnose disease, e.g.,immune-compromised conditions, monocyte depleted conditions, oroverproduction of monocyte.

[0098] Antibodies raised against each monocyte will also be useful toraise anti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

[0099] b. Immunoassays

[0100] A particular protein can be measured by a variety of immunoassaymethods. For a review of immunological and immunoassay procedures ingeneral, see Stites and Terr (eds.) 1991 Basic and Clinical Immunology(7th ed.). Moreover, the immunoassays of the present invention can beperformed in any of several configurations, which are reviewedextensively in Maggio (ed.) (1980) Enzyme Immunoassay CRC Press, BocaRaton, Fla.; Tijan (1985) “Practice and Theory of Enzyme Immunoassays,”Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers B.V., Amsterdam; and Harlow and Lane Antibodies, ALaboratory Manual, supra, each of which is incorporated herein byreference. See also Chan (ed.) (1987) Immunoassay: A Practical GuideAcademic Press, Orlando, Fla.; Price and Newman (eds.) (1991) Principlesand Practice of Immunoassays Stockton Press, NY; and Ngo (ed.) (1988)Non-isotopic Immunoassays Plenum Press, NY.

[0101] Immunoassays for measurement of these monocyte proteins can beperformed by a variety of methods known to those skilled in the art. Inbrief, immunoassays to measure the protein can be competitive ornoncompetitive binding assays. In competitive binding assays, the sampleto be analyzed competes with a labeled analyte for specific bindingsites on a capture agent bound to a solid surface. Preferably thecapture agent is an antibody specifically reactive with the monocyteprotein produced as described above. The concentration of labeledanalyte bound to the capture agent is inversely proportional to theamount of free analyte present in the sample.

[0102] In a competitive binding immunoassay, the monocyte proteinpresent in the sample competes with labeled protein for binding to aspecific binding agent, for example, an antibody specifically reactivewith the monocyte protein. The binding agent may be bound to a solidsurface to effect separation of bound labeled protein from the unboundlabeled protein. Alternately, the competitive binding assay may beconducted in liquid phase and any of a variety of techniques known inthe art may be used to separate the bound labeled protein from theunbound labeled protein. Following separation, the amount of boundlabeled protein is determined. The amount of protein present in thesample is inversely proportional to the amount of labeled proteinbinding.

[0103] Alternatively, a homogeneous immunoassay may be performed inwhich a separation step is not needed. In these immunoassays, the labelon the protein is altered by the binding of the protein to its specificbinding agent. This alteration in the labeled protein results in adecrease or increase in the signal emitted by label, so that measurementof the label at the end of the immunoassay allows for detection orquantitation of the protein.

[0104] These monocyte proteins may also be quantitatively determined bya variety of noncompetitive immunoassay methods. For example, atwo-site, solid phase sandwich immunoassay may be used. In this type ofassay, a binding agent for the protein, for example an antibody, isattached to a solid support. A second protein binding agent, which mayalso be an antibody, and which binds the protein at a different site, islabeled. After binding at both sites on the protein has occurred, theunbound labeled binding agent is removed and the amount of labeledbinding agent bound to the solid phase is measured. The amount oflabeled binding agent bound is directly proportional to the amount ofprotein in the sample.

[0105] Western blot analysis can be used to determine the presence ofmonocyte proteins in a sample. Electrophoresis is carried out, e.g., ona tissue sample suspected of containing the protein. Followingelectrophoresis to separate the proteins, and transfer of the proteinsto a suitable solid support such as a nitrocellulose filter, the solidsupport is incubated with an antibody reactive with the denaturedprotein. This antibody may be labeled, or alternatively may be it may bedetected by subsequent incubation with a second labeled antibody thatbinds the primary antibody.

[0106] The immunoassay formats described above employ labeled assaycomponents. The label can be in a variety of forms. The label may becoupled directly or indirectly to the desired component of the assayaccording to methods well known in the art. A wide variety of labels maybe used. The component may be labeled by any one of several methods.Traditionally a radioactive label incorporating ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P is used. Non-radioactive labels include ligands which bind tolabeled antibodies, fluorophores, chemiluminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabeled protein. The choice of label depends on sensitivity required,ease of conjugation with the compound, stability requirements, andavailable instrumentation. For a review of various labeling or signalproducing systems which may be used, see U.S. Pat. No. 4,391,904, whichis incorporated herein by reference.

[0107] Antibodies reactive with a particular protein can also bemeasured by a variety of immunoassay methods. For reviews ofimmunological and immunoassay procedures applicable to the measurementof antibodies by immunoassay techniques, see, e.g., Stites and Terr(eds.) Basic and Clinical Immunology (7th ed.) supra; Maggio (ed.)Enzyme Immunoassay, supra; and Harlow and Lane Antibodies, A LaboratoryManual, supra.

[0108] A variety of different immunoassay formats, separationtechniques, and labels can be also be used similar to those describedabove for the measurement of specific proteins. Moreover, many methodsare known for evaluating selectivity of binding for specific protein orclosely related proteins.

[0109] VI. Purified Monocyte Proteins

[0110] The human monocyte FDF03 protein amino acid sequence is providedin SEQ ID NO: 2. Partial mouse sequence is provided in SEQ ID NO: 4.Human YE01 amino acid and nucleotide sequences for the Ig-family memberare provided in SEQ ID NO: 5-10. The receptor family members, designatedKTE03, including the YYB01, YYB04, and KLM63, KLM66, and KLM67embodiments, are described in SEQ ID NO: 11-22.

[0111] The peptide sequences allow preparation of peptides to generateantibodies to recognize such segments, and allow preparation ofoligonucleotides which encode such sequences. Moreover, affinityreagents allow detection and purification of more protein, includingfull length or recombinant forms. And oligonucleotide sequences allowdetection of cDNAs encoding, or closely related to, these.

[0112] VII. Physical Variants

[0113] This invention also encompasses proteins or peptides havingsubstantial amino acid sequence similarity with an amino acid sequenceof SEQ ID NO: 2 or 4; 6, 8, or 10; or 12, 14, 16, 18, 20, or 22,especially splice variants. Variants exhibiting substitutions, e.g., 20or fewer, preferably 10 or fewer, and more preferably 5 or fewersubstitutions, are also enabled. Where the substitutions areconservative substitutions, the variants will share immunogenic orantigenic similarity or cross-reactivity with a corresponding naturalsequence protein. Natural variants include individual, allelic,polymorphic, strain, or species variants.

[0114] Amino acid sequence similarity, or sequence identity, isdetermined by optimizing residue matches, if necessary, by introducinggaps as required. This changes when considering conservativesubstitutions as matches. Conservative substitutions typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. Homologous amino acid sequences include natural allelic andinterspecies variations in each respective protein sequence. Typicalhomologous proteins or peptides will have from 50-100% similarity (ifgaps can be introduced), to 75-100% similarity (if conservativesubstitutions are included) with the amino acid sequence of the relevantmonocyte protein. Identity measures will be at least about 50%,generally at least 60%, more generally at least 65%, usually at least70%, more usually at least 75%, preferably at least 80%, and morepreferably at least 80%, and in particularly preferred embodiments, atleast 85% or more. See also Needleham, et al. (1970) J. Mol. Biol.48:443-453; Sankoff, et al. (1983) Time Warps, String Edits, andMacromolecules: The Theory and Practice of Seauence Comparison ChapterOne, Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group (GCG), Madison, Wis.

[0115] Nucleic acids encoding the corresponding mammalian monocyteproteins will typically hybridize, e.g., to SEQ ID NO 1 and/or 3; 5, 7,and/or 9; or 11, 13, 15, 17, 19, and/or 21 under stringent conditions.For example, nucleic acids encoding the respective monocyte proteinswill typically hybridize to the appropriate nucleic acid under stringenthybridization conditions, while providing few false positivehybridization signals. Generally, stringent conditions are selected tobe about 10° C. lower than the thermal melting point (Tm) for thesequence being hybridized to at a defined ionic strength and pH. The Tmis the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration inwash is about 0.02 molar at pH 7 and the temperature is at least about50° C. Other factors may significantly affect the stringency ofhybridization, including, among others, base composition and size of thecomplementary strands, the presence of organic solvents such asformamide, and the extent of base mismatching. A preferred embodimentwill include nucleic acids which will bind to disclosed sequences in 50%formamide and 20-50 mM NaCl at 42° C. In certain cases, the stringencymay be relaxed to detect other nucleic acids exhibiting less thancomplete sequence identity.

[0116] An isolated monocyte gene DNA can be readily modified bynucleotide substitutions, nucleotide deletions, nucleotide insertions,and inversions of nucleotide stretches. These modifications result innovel DNA sequences which encode these monocyte antigens, theirderivatives, or proteins having highly similar physiological,immunogenic, or antigenic activity.

[0117] Modified sequences can be used to produce mutant antigens or toenhance expression. Enhanced expression may involve gene amplification,increased transcription, increased translation, and other mechanisms.Such mutant monocyte protein derivatives include predetermined orsite-specific mutations of the respective protein or its fragments.“Mutant monocyte protein” encompasses a polypeptide otherwise fallingwithin the homology definition of the monocyte protein as set forthabove, but having an amino acid sequence which differs from that of themonocyte protein as found in nature, whether by way of deletion,substitution, or insertion. In particular, “site specific mutantmonocyte protein” generally includes proteins having significantsimilarity with a protein having a sequence of SEQ ID NO: 2 or 4; 6, 8,or 10; or 12, 14, 16, 18, 20, or 22. Generally, the variant will sharemany physicochemical and biological activities, e.g., antigenic orimmunogenic, with those sequences, and in preferred embodiments containmost or all of the disclosed sequence. Similar concepts apply to thesevarious monocyte proteins, particularly those found in various warmblooded animals, e.g., primates and mammals.

[0118] Although site specific mutation sites are predetermined, mutantsneed not be site specific. Monocyte protein mutagenesis can be conductedby making amino acid insertions or deletions. Substitutions, deletions,insertions, or any combinations may be generated to arrive at a finalconstruct. Insertions include amino- or carboxyl-terminal fusions.Random mutagenesis can be conducted at a target codon and the expressedmutants can then be screened for the desired activity. Methods formaking substitution mutations at predetermined sites in DNA having aknown sequence are well known in the art, e.g., by M13 primermutagenesis or polymerase chain reaction (PCR) techniques. See also,Sambrook, et al. (1989) and Ausubel, et al. (1987 and Supplements). Themutations in the DNA normally should not place coding sequences out ofreading frames and preferably will not create complementary regions thatcould hybridize to produce secondary mRNA structure such as loops orhairpins.

[0119] The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. Thus, the fusionproduct of an immunoglobulin with a respective monocyte polypeptide is acontinuous protein molecule having sequences fused in a typical peptidelinkage, typically made as a single translation product and exhibitingproperties derived from each source peptide. A similar concept appliesto heterologous nucleic acid sequences.

[0120] In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, domains or othersegments may be “swapped” between different new fusion polypeptides orfragments, typically with related proteins, e.g., within the Ig familyor the Fc receptor family. Preferably, intact structural domains will beused, e.g., intact Ig portions. See, e.g., Cunningham, et al. (1989)Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem.263:15985-15992. Thus, new chimeric polypeptides exhibiting newcombinations of specificities will result from the functional linkage ofprotein-binding specificities and other functional domains. Also,alanine scanning mutagenesis may be applied, preferably to residueswhich structurally are exterior to the secondary structure, which willavoid most of the critical residues which generally disrupt tertiarystructure.

[0121] “Derivatives” of these monocyte antigens include amino acidsequence mutants, glycosylation variants, and covalent or aggregateconjugates with other chemical moieties. Covalent derivatives can beprepared by linkage of functionalities to groups which are found inthese monocyte protein amino acid side chains or at the N- or C-termini,by means which are well known in the art. These derivatives can include,without limitation, aliphatic esters or amides of the carboxyl terminus,or of residues containing carboxyl side chains, O-acyl derivatives ofhydroxyl group-containing residues, and N-acyl derivatives of the aminoterminal amino acid or amino-group containing residues, e.g., lysine orarginine. Acyl groups are selected from the group of alkyl-moietiesincluding C3 to C18 normal alkyl, thereby forming alkanoyl aroylspecies. Covalent attachment to carrier proteins may be important whenimmunogenic moieties are haptens.

[0122] In particular, glycosylation alterations are included, e.g., madeby modifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., mammalian glycosylation enzymes. Deglycosylationenzymes are also contemplated. Also embraced are versions of the sameprimary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine, or other moieties, including ribosylgroups or cross-linking reagents. Also, proteins comprisingsubstitutions are encompassed, which should retain substantialimmunogenicity, to produce antibodies which recognize a protein of SEQID NO: 2 or 4; 6, 8, or 10; or 12, 14, 16, 18, 20, or 22. Alternatively,it may be desired to produce antibodies which recognize all or subsetsof SEQ ID NO: 2 and 4; 6, 8, and 10; or 12, 14, 16, 18, 20, and 22.Typically, these proteins will contain less than 20 residuesubstitutions from the disclosed sequence, more typically less than 10substitutions, preferably less than 5, and more preferably less than 3.Alternatively, proteins which begin and end at structural domains willusually retain antigenicity and cross immunogenicity.

[0123] A major group of derivatives are covalent conjugates of themonocyte proteins or fragments thereof with other proteins orpolypeptides. These derivatives can be synthesized in recombinantculture such as N- or C-terminal fusions or by the use of agents knownin the art for their usefulness in cross-linking proteins throughreactive side groups. Preferred protein derivatization sites withcross-linking agents are at free amino groups, carbohydrate moieties,and cysteine residues.

[0124] Fusion polypeptides between these monocyte proteins and otherhomologous or heterologous proteins are also provided. Heterologouspolypeptides may be fusions between different surface markers, resultingin, e.g., a hybrid protein. Likewise, heterologous fusions may beconstructed which would exhibit a combination of properties oractivities of the derivative proteins. Typical examples are fusions of areporter polypeptide, e.g., luciferase, with a segment or domain of aprotein, e.g., a receptor-binding segment, so that the presence orlocation of the fused protein may be easily determined. See, e.g., Dull,et al., U.S. Pat. No. 4,859,609. Other gene fusion partners includebacterial β-galactosidase, trpE, Protein A, β-lactamase, alpha amylase,alcohol dehydrogenase, and yeast alpha mating factor. See, e.g.,Godowski, et al. (1988) Science 241:812-816.

[0125] Such polypeptides may also have amino acid residues which havebeen chemically modified by phosphorylation, sulfonation, biotinylation,or the addition or removal of other moieties, particularly those whichhave molecular shapes similar to phosphate groups. In some embodiments,the modifications will be useful labeling reagents, or serve aspurification targets, e.g., affinity ligands.

[0126] This invention also contemplates the use of derivatives of thesemonocyte proteins other than variations in amino acid sequence orglycosylation. Such derivatives may involve covalent or aggregativeassociation with chemical moieties. These derivatives generally fallinto the three classes: (1) salts, (2) side chain and terminal residuecovalent modifications, and (3) adsorption complexes, for example withcell membranes. Such covalent or aggregative derivatives are useful asimmunogens, as reagents in immunoassays, or in purification methods suchas for affinity purification of ligands or other binding ligands. Forexample, a monocyte protein antigen can be immobilized by covalentbonding to a solid support such as cyanogen bromide-activated Sepharose,by methods which are well known in the art, or adsorbed onto polyolefinsurfaces, with or without glutaraldehyde cross-linking, for use in theassay or purification of anti-monocyte protein antibodies. The monocyteproteins can also be labeled with a detectable group, e.g.,radioiodinated by the chloramine T procedure, covalently bound to rareearth chelates, or conjugated to another fluorescent moiety for use indiagnostic assays. Purification of these monocyte proteins may beeffected by immobilized antibodies.

[0127] Isolated monocyte protein genes will allow transformation ofcells lacking expression of a corresponding monocyte protein, e.g.,either species types or cells which lack corresponding proteins andexhibit negative background activity. Expression of transformed geneswill allow isolation of antigenically pure cell lines, with defined orsingle specie variants. This approach will allow for more sensitivedetection and discrimination of the physiological effects of thesemonocyte proteins. Subcellular fragments, e.g., cytoplasts or membranefragments, can be isolated and used.

[0128] VIII. Binding Agent: Monocyte Protein Complexes

[0129] A monocyte protein that specifically binds to or that isspecifically immunoreactive with an antibody generated against a definedimmunogen, such as an immunogen consisting of the amino acid sequence ofSEQ ID NO: 2 and/or 4; 6, 8, and/or 10; or 12, 14, 16, 18, 20, and/or22, is determined in an immunoassay. The immunoassay uses a polyclonalantiserum which was raised to the protein of SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, or 22, or appropriate combination. This antiserum isselected to have low crossreactivity against other members of therelated families, and any such crossreactivity is, or may be, removed byimmunoabsorption prior to use in the immunoassay.

[0130] In order to produce antisera for use in an immunoassay, theprotein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22 isisolated as described herein. For example, recombinant protein may beproduced in a mammalian cell line. An inbred strain of mice such asBalb/c is immunized with the appropriate protein using a standardadjuvant, such as Freund's adjuvant, and a standard mouse immunizationprotocol (see Harlow and Lane, supra). Alternatively, a syntheticpeptide derived from the sequences disclosed herein and conjugated to acarrier protein can be used an immunogen. Polyclonal sera are collectedand titered against the immunogen protein in an immunoassay, e.g., asolid phase immunoassay with the immunogen immobilized on a solidsupport. Polyclonal antisera with a titer of 104 or greater are selectedand tested for their cross reactivity against other related proteins,using a competitive binding immunoassay such as the one described inHarlow and Lane, supra, at pages 570-573. See also Hertzenberg, et al.(eds. 1996) Weir's Handbook of Experimental Immunology vols. 1-4,Blackwell Science; and Coligan (1991) Current Protocols in ImmunologyWiley/Greene, NY. Preferably two different related proteins are used inthis determination in conjunction with a given monocyte protein. Forexample, with the Ig family protein, at least two other family membersare used to absorb out shared epitopes. In conjunction with the Fcfamily member, two other members of the family are used. These otherfamily members can be produced as recombinant proteins and isolatedusing standard molecular biology and protein chemistry techniques asdescribed herein.

[0131] Immunoassays in the competitive binding format can be used forthe crossreactivity determinations. For example, the protein can beimmobilized to a solid support. Proteins added to the assay compete withthe binding of the antisera to the immobilized antigen. The ability ofthe above proteins to compete with the binding of the antisera to theimmobilized protein is compared to the protein of SEQ ID NO 2 and/or 4;6, 8, and/or 10; or 12, 14, 16, 18, 20, and/or 22. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the proteins listed above are selected and pooled. Thecross-reacting antibodies are then removed from the pooled antisera byimmunoabsorption with the above-listed proteins.

[0132] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described above to compare a secondprotein to the immunogen protein, e.g., the monocyte protein of SEQ IDNO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22. In order to make thiscomparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of the second protein required is less than twice the amountof the protein, e.g., of SEQ ID NO: 2, that is required, then the secondprotein is said to specifically bind to an antibody generated to theimmunogen.

[0133] It is understood that monocyte proteins are each a family ofhomologous proteins that comprise two or more genes. For a particulargene product, such as the human Ig family member protein, the inventionencompasses not only the amino acid sequences disclosed herein, but alsoto other proteins that are allelic, polymorphic, non-allelic, or speciesvariants. It is also understood that the term “human monocyte protein”includes normatural mutations introduced by deliberate mutation usingconventional recombinant technology such as single site mutation, or byexcising short sections of DNA encoding these proteins or splicevariants from the gene, or by substituting or adding small numbers ofnew amino acids. Such minor alterations must substantially maintain theimmunoidentity of the original molecule and/or its biological activity.Thus, these alterations include proteins that are specificallyimmunoreactive with a designated naturally occurring respective monocyteprotein, for example, the human monocyte protein exhibiting SEQ ID NO:4. Particular protein modifications considered minor would includeconservative substitution of amino acids with similar chemicalproperties, as described above for each protein family as a whole. Byaligning a protein optimally with the protein of SEQ ID NO 2 and 4; 6,8, and 10; or 12, 14, 16, 18, 20, and 22, and by using the conventionalimmunoassays described herein to determine immunoidentity, one candetermine the protein compositions of the invention.

[0134] IX. Uses

[0135] The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in thegeneral description for developmental abnormalities, or below in thedescription of kits for diagnosis.

[0136] Monocyte genes, e.g., DNA or RNA may be used as a component in aforensic assay. For instance, the nucleotide sequences provided may belabeled using, e.g., ³²P or biotin and used to probe standardrestriction fragment polymorphism blots, providing a measurablecharacter to aid in distinguishing between individuals. Such probes maybe used in well-known forensic techniques such as geneticfingerprinting. In addition, nucleotide probes made from monocytesequences may be used in in situ assays to detect chromosomalabnormalities.

[0137] Antibodies and other binding agents directed towards monocyteproteins or nucleic acids may be used to purify the correspondingmonocyte protein molecule. As described in the Examples below, antibodypurification of monocyte proteins is both possible and practicable.Antibodies and other binding agents may also be used in a diagnosticfashion to determine whether monocyte components are present in a tissuesample or cell population using well-known techniques described herein.The ability to attach a binding agent to a monocyte protein provides ameans to diagnose disorders associated with expression misregulation.Antibodies and other monocyte protein binding agents may also be usefulas histological markers. As described in the examples below, theexpression of each of these proteins is limited to specific tissuetypes. By directing a probe, such as an antibody or nucleic acid to therespective monocyte protein, it is possible to use the probe todistinguish tissue and cell types in situ or in vitro.

[0138] This invention also provides reagents which may exhibitsignificant therapeutic value. The monocyte proteins (naturallyoccurring or recombinant), fragments thereof, and antibodies thereto,along with compounds identified as having binding affinity to themonocyte protein, may be useful in the treatment of conditionsassociated with abnormal physiology or development, including abnormalproliferation, e.g., cancerous conditions, or degenerative conditions.Abnormal proliferation, regeneration, degeneration, and atrophy may bemodulated by appropriate therapeutic treatment using the compositionsprovided herein. For example, a disease or disorder associated withabnormal expression or abnormal signaling by a monocyte, e.g., as anantigen presenting cell, is a target for an agonist or antagonist of theprotein. The proteins likely play a role in regulation or development ofhematopoietic cells, e.g., lymphoid cells, which affect immunologicalresponses, e.g., antigen presentation and the resulting effectorfunctions.

[0139] For example, the DX26 antibody shows that inhibitory antibodieswill be useful in modulating NK or T cell functions, e.g., killing. Suchmodulation will typically be a 20% effect, either increasing ordecreasing, e.g., the killing effect, but in preferred embodiments willhave a 30%, 40%, 50%, or more. Because the distribution is also inmonocytes, the molecule will probably also affect the regulation ofmonocyte mediated or initiated effector functions of the immune system,e.g., autoimmune responses, transplantation rejection, graft vs. hostdisease, inflammatory conditions, etc. These molecules may also affectelimination of neoplastic conditions, e.g., tumor rejection.

[0140] Other abnormal developmental conditions are known in cell typesshown to possess monocyte protein mRNA by northern blot analysis. SeeBerkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co.,Rahway, N.J.; and Thorn, et al. Harrison's Principles of InternalMedicine, McGraw-Hill, NY. Developmental or functional abnormalities,e.g., of the immune system, cause significant medical abnormalities andconditions which may be susceptible to prevention or treatment usingcompositions provided herein.

[0141] Recombinant monocyte proteins or antibodies might be purified andthen administered to a patient. These reagents can be combined fortherapeutic use with additional active or inert ingredients, e.g., inconventional pharmaceutically acceptable carriers or diluents, e.g.,immunogenic adjuvants, along with physiologically innocuous stabilizersand excipients. In particular, these may be useful in a vaccine context,where the antigen is combined with one of these therapeutic versions ofagonists or antagonists. These combinations can be sterile filtered andplaced into dosage forms as by lyophilization in dosage vials or storagein stabilized aqueous preparations. This invention also contemplates useof antibodies or binding fragments thereof, including forms which arenot complement binding.

[0142] Drug screening using antibodies or receptor or fragments thereofcan identify compounds having binding affinity to these monocyteproteins, including isolation of associated components. Subsequentbiological assays can then be utilized to determine if the compound hasintrinsic stimulating activity and is therefore a blocker or antagonistin that it blocks the activity of the protein. Likewise, a compoundhaving intrinsic stimulating activity might activate the cell throughthe protein and is thus an agonist in that it simulates the cell. Thisinvention further contemplates the therapeutic use of antibodies to theproteins as antagonists.

[0143] The quantities of reagents necessary for effective therapy willdepend upon many different factors, including means of administration,target site, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics(8th ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 μM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous administration.

[0144] The monocyte proteins, fragments thereof, and antibodies to it orits fragments, antagonists, and agonists, could be administered directlyto the host to be treated or, depending on the size of the compounds, itmay be desirable to conjugate them to carrier proteins such as ovalbuminor serum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990)Goodman and Gilman's: The Pharmacoloaical Bases of Therapeutics (8thed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa.; Avis, et al. (eds.) (1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: TabletsDekker, NY; and Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Disperse Systems Dekker, NY. The therapy of this invention may becombined with or used in association with other chemotherapeutic orchemopreventive agents.

[0145] Both the naturally occurring and the recombinant form of themonocyte proteins of this invention are particularly useful in kits andassay methods which are capable of screening compounds for bindingactivity to the proteins. Several methods of automating assays have beendeveloped in recent years so as to permit screening of tens of thousandsof compounds in a short period. See, e.g., Fodor, et al. (1991) Science251:767-773, and other descriptions of chemical diversity libraries,which describe means for testing of binding affinity by a plurality ofcompounds. The development of suitable assays can be greatly facilitatedby the availability of large amounts of purified, e.g., soluble versionsof, monocyte protein as provided by this invention.

[0146] For example, antagonists can often be found once the protein hasbeen structurally defined. Testing of potential protein analogs is nowpossible upon the development of highly automated assay methods using apurified surface protein. In particular, new agonists and antagonistswill be discovered by using screening techniques described herein. Ofparticular importance are compounds found to have a combined bindingaffinity for multiple related cell surface antigens, e.g., compoundswhich can serve as antagonists for species variants of a monocyteprotein.

[0147] This invention is particularly useful for screening compounds byusing recombinant monocyte protein in a variety of drug screeningtechniques. The advantages of using a recombinant protein in screeningfor specific ligands include: (a) improved renewable source of theprotein from a specific source; (b) potentially greater number ofantigens per cell giving better signal to noise ratio in assays; and (c)species variant specificity (theoretically giving greater biological anddisease specificity).

[0148] One method of drug screening utilizes eukaryotic or prokaryotichost cells which are stably transformed with recombinant DNA moleculesexpressing a monocyte protein. Cells may be isolated which express thatprotein in isolation from any others. Such cells, either in viable orfixed form, can be used for standard surface protein binding assays. Seealso, Parce, et al. (1989) Science 246:243-247; and Owicki, et al.(1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses. Competitive assays areparticularly useful, where the cells (source of monocyte protein) arecontacted and incubated with an antibody having known binding affinityto the antigen, such as ¹²⁵I-antibody, and a test sample whose bindingaffinity to the binding composition is being measured. The bound andfree labeled binding compositions are then separated to assess thedegree of protein binding. The amount of test compound bound isinversely proportional to the amount of labeled antibody binding to theknown source. Many techniques can be used to separate bound from freereagent to assess the degree of binding. This separation step couldtypically involve a procedure such as adhesion to filters followed bywashing, adhesion to plastic followed by washing, or centrifugation ofthe cell membranes. Viable cells could also be used to screen for theeffects of drugs on these monocyte protein mediated functions, e.g.,antigen presentation or helper function.

[0149] Another method utilizes membranes from transformed eukaryotic orprokaryotic host cells as the source of a monocyte protein. These cellsare stably transformed with DNA vectors directing the expression of theappropriate protein, e.g., an engineered membrane bound form.Essentially, the membranes would be prepared from the cells and used inbinding assays such as the competitive assay set forth above.

[0150] Still another approach is to use solubilized, unpurified orsolubilized, purified monocyte protein from transformed eukaryotic orprokaryotic host cells. This allows for a “molecular” binding assay withthe advantages of increased specificity, the ability to automate, andhigh drug test throughput.

[0151] Another technique for drug screening involves an approach whichprovides high throughput screening for compounds having suitable bindingaffinity to the respective monocyte protein and is described in detailin Geysen, European Patent Application 84/03564, published on Sep. 13,1984. First, large numbers of different small peptide test compounds aresynthesized on a solid substrate, e.g., plastic pins or some otherappropriate surface, see Fodor, et al., supra. Then all the pins arereacted with solubilized, unpurified or solubilized, purified monocyteprotein, and washed. The next step involves detecting bound reagent,e.g., antibody.

[0152] One means for determining which sites interact with specificother proteins is a physical structure determination, e.g., x-raycrystallography or 2 dimensional NMR techniques. These will provideguidance as to which amino acid residues form molecular contact regions.For a detailed description of protein structural determination, see,e.g., Blundell and Johnson (1976) Protein Crystallography AcademicPress, NY.

[0153] X. Kits

[0154] This invention also contemplates use of these monocyte proteins,fragments thereof, peptides, and their fusion products in a variety ofdiagnostic kits and methods for detecting the presence of a monocyteprotein or message. Typically the kit will have a compartment containingeither a defined monocyte peptide or gene segment or a reagent whichrecognizes one or the other, e.g., antibodies.

[0155] A kit for determining the binding affinity of a test compound tothe respective monocyte protein would typically comprise a testcompound; a labeled compound, for example an antibody having knownbinding affinity for the protein; a source of the monocyte protein(naturally occurring or recombinant); and a means for separating boundfrom free labeled compound, such as a solid phase for immobilizing themonocyte protein. Once compounds are screened, those having suitablebinding affinity to the protein can be evaluated in suitable biologicalassays, as are well known in the art, to determine whether they act asagonists or antagonists to regulate monocyte function. The availabilityof recombinant monocyte polypeptides also provide well defined standardsfor calibrating such assays.

[0156] A preferred kit for determining the concentration of, forexample, a monocyte protein in a sample would typically comprise alabeled compound, e.g., antibody, having known binding affinity for themonocyte protein, a source of monocyte protein (naturally occurring orrecombinant) and a means for separating the bound from free labeledcompound, for example, a solid phase for immobilizing the monocyteprotein. Compartments containing reagents, and instructions, willnormally be provided.

[0157] Antibodies, including antigen binding fragments, specific for therespective monocyte or its fragments are useful in diagnosticapplications to detect the presence of elevated levels of the proteinand/or its fragments. Such diagnostic assays can employ lysates, livecells, fixed cells, immunofluorescence, cell cultures, body fluids, andfurther can involve the detection of antigens in serum, or the like.Diagnostic assays may be homogeneous (without a separation step betweenfree reagent and antigen-monocyte protein complex) or heterogeneous(with a separation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like.For example, unlabeled antibodies can be employed by using a secondantibody which is labeled and which recognizes the antibody to themonocyte protein or to a particular fragment thereof. Similar assayshave also been extensively discussed in the literature. See, e.g.,Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press, NY;Chan (ed.) (1987) Immunoassay: A Practical Guide Academic Press,Orlando, Fla.; Price and Newman (eds.) (1991) Principles and Practice ofImmunoassay Stockton Press, NY; and Ngo (ed.) (1988) NonisotopicImmunoassay Plenum Press, NY. In particular, the reagents may be usefulfor diagnosing monocyte populations in biological samples, either todetect an excess or deficiency of monocyte in a sample. The assay may bedirected to histological analysis of a biopsy, or evaluation of monocytenumbers in a blood or tissue sample.

[0158] Anti-idiotypic antibodies may have similar use to diagnosepresence of antibodies against a monocyte protein, as such may bediagnostic of various abnormal states. For example, overproduction ofthe monocyte protein may result in various immunological reactions whichmay be diagnostic of abnormal physiological states, particularly inproliferative cell conditions such as cancer or abnormaldifferentiation.

[0159] Frequently, the reagents for diagnostic assays are supplied inkits, so as to optimize the sensitivity of the assay. For the subjectinvention, depending upon the nature of the assay, the protocol, and thelabel, either labeled or unlabeled antibody or receptor, or labeledmonocyte protein is provided. This is usually in conjunction with otheradditives, such as buffers, stabilizers, materials necessary for signalproduction such as substrates for enzymes, and the like. Preferably, thekit will also contain instructions for proper use and disposal of thecontents after use. Typically the kit has compartments for each usefulreagent. Desirably, the reagents are provided as a dry lyophilizedpowder, where the reagents may be reconstituted in an aqueous mediumproviding appropriate concentrations of reagents for performing theassay.

[0160] Many of the aforementioned constituents of the drug screening andthe diagnostic assays may be used without modification or may bemodified in a variety of ways. For example, labeling may be achieved bycovalently or non-covalently joining a moiety which directly orindirectly provides a detectable signal. In many of these assays, theprotein, test compound, monocyte protein, or antibodies thereto can belabeled either directly or indirectly. Possibilities for direct labelinginclude label groups: radiolabels such as ¹²⁵I, enzymes (U.S. Pat. No.3,645,090) such as peroxidase and alkaline phosphatase, and fluorescentlabels (U.S. Pat. No. 3,940,475) capable of monitoring the change influorescence intensity, wavelength shift, or fluorescence polarization.Possibilities for indirect labeling include biotinylation of oneconstituent followed by binding to avidin coupled to one of the abovelabel groups.

[0161] There are also numerous methods of separating the bound from thefree protein, or alternatively the bound from the free test compound.The monocyte protein can be immobilized on various matrices followed bywashing. Suitable matrices include plastic such as an ELISA plate,filters, and beads. Methods of immobilizing the monocyte protein to amatrix include, without limitation, direct adhesion to plastic, use of acapture antibody, chemical coupling, and biotin-avidin. The last step inthis approach involves the precipitation of protein/antibody complex byone of several methods including those utilizing, e.g., an organicsolvent such as polyethylene glycol or a salt such as ammonium sulfate.Other suitable separation techniques include, without limitation, thefluorescein antibody magnetizable particle method described in Rattle,et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magneticparticle separation as described in U.S. Pat. No. 4,659,678.

[0162] Methods for linking proteins or their fragments to the variouslabels have been extensively reported in the literature and do notrequire detailed discussion here. Many of the techniques involve the useof activated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

[0163] Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence of arespective monocyte protein. These sequences can be used as probes fordetecting levels of the message in samples from patients suspected ofhaving an abnormal condition, e.g., cancer or immune problem. Thepreparation of both RNA and DNA nucleotide sequences, the labeling ofthe sequences, and the preferred size of the sequences has receivedample description and discussion in the literature. Normally anoligonucleotide probe should have at least about 14 nucleotides, usuallyat least about 18 nucleotides, and the polynucleotide probes may be upto several kilobases. Various labels may be employed, most commonlyradionuclides, particularly ³²P. However, other techniques may also beemployed, such as using biotin modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorophores, enzymes, or the like.Alternatively, antibodies may be employed which can recognize specificduplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes,or DNA-protein duplexes. The antibodies in turn may be labeled and theassay carried out where the duplex is bound to a surface, so that uponthe formation of duplex on the surface, the presence of antibody boundto the duplex can be detected. The use of probes to the novel anti-senseRNA may be carried out in any conventional techniques such as nucleicacid hybridization, plus and minus screening, recombinational probing,hybrid released translation (HRT), and hybrid arrested translation(HART). This also includes amplification techniques such as polymerasechain reaction (PCR).

[0164] Diagnostic kits which also test for the qualitative orquantitative presence of other markers are also contemplated. Diagnosisor prognosis may depend on the combination of multiple indications usedas markers. Thus, kits may test for combinations of markers. See, e.g.,Viallet, et al. (1989) Progress in Growth Factor Res. 1:89-97.

[0165] XI. Binding Partner Isolation

[0166] Having isolated one member of a binding partner of a specificinteraction, methods exist for isolating the counter-partner. See,Gearing, et al. (1989) EMBO J. 8:3667-3676. For example, means to labela monocyte surface protein without interfering with the binding to itsreceptor can be determined. For example, an affinity label can be fusedto either the amino- or carboxyl-terminus of the ligand. An expressionlibrary can be screened for specific binding to the monocyte protein,e.g., by cell sorting, or other screening to detect subpopulations whichexpress such a binding component. See, e.g., Ho, et al. (1993) Proc.Nat'l Acad. Sci. USA 90:11267-11271. Alternatively, a panning method maybe used. See, e.g., Seed and Aruffo (1987) Proc. Nat'l Acad. Sci. USA84:3365-3369. A two-hybrid selection system may also be applied makingappropriate constructs with the available monocyte protein sequences.See, e.g., Fields and Song (1989) Nature 340:245-246.

[0167] Protein cross-linking techniques with label can be applied toisolate binding partners of a monocyte protein. This would allowidentification of proteins which specifically interact with theappropriate monocyte protein.

[0168] The broad scope of this invention is best understood withreference to the following examples, which are not intended to limit theinvention to specific embodiments.

EXAMPLES

[0169] I. General Methods

[0170] Many of the standard methods below are described or referenced,e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory ManualCold Spring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook,et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1-3,CSH Press, NY; Ausubel, et al., Biology Greene Publishing Associates,Brooklyn, NY; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology Wiley/Greene, NY; Innis, et al. (eds.)(1990) PCR Protocols: A Guide to Methods and Applications AcademicPress, NY.

[0171] Methods for protein purification include such methods as ammoniumsulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification,” Methods in Enzmmology vol. 182, and other volumes in thisseries; Coligan, et al. (1996 and periodic Supplements) CurrentProtocols in Protein Science Wiley/Greene, NY; and manufacturer'sliterature on use of protein purification products, e.g., Pharmacia,Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combination withrecombinant techniques allow fusion to appropriate segments, e.g., to aFLAG sequence or an equivalent which can be fused via aprotease-removable sequence. See, e.g., Hochuli (1989) ChemischeIndustrie 12:69-70; Hochuli (1990) “Purification of Recombinant Proteinswith Metal Chelate Absorbent” in Setlow (ed.) Genetic Engineering,Principle and Methods 12:87-98, Plenum Press, NY; and Crowe, et al.(1992) OIAexpress: The High Level Expression & Protein PurificationSystem QUIAGEN, Inc., Chatsworth, Calif.

[0172] Standard immunological techniques are described, e.g., inHertzenberg, et al. (eds. 1996) Weir's Handbook of ExperimentalImmunology vols. 1-4, Blackwell Science; Coligan (1991) CurrentProtocols in Immunology Wiley/Greene, NY; and Methods in Enzmmologyvolumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163.See also, e.g., Paul (ed.) (1993) Fundamental Immunology (3d ed.) RavenPress, NY.

[0173] FACS analyses are described in Melamed, et al. (1990) FlowCytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988)Practical Flow Cytometry Liss, New York, N.Y.; and Robinson, et al.(1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y.

[0174] II. Isolation of Human Monocytes

[0175] Healthy donors were subjected to a leukophoresis. Percollgradients were used to isolate mononuclear cells which were then subjectto centrifugal elutriation. See, Figdor, et al. (1982) Blood 60:46-53;and Plas, et al. (1988) Expt'l. Hematol. 16:355-359. This highlyenriched monocyte fraction was cultured for 5-7 days in the presence ofGM-CSF (800 U/ml) and IL-4 (500 U/ml), as described in Romani, et al(1994) J. Exp. Med. 180:83-93; and Sallusto, et al (1994) J. Exp. Med.179:1109-1118.

[0176] For making dendritic cells, human CD34+ cells were obtained asfollows. See, e.g., Caux, et al. (1995) pages 1-5 in Banchereau andSchmitt Dendritic Cells in Fundamental and Clinical Immunology PlenumPress, NY. Peripheral or cord blood cells, sometimes CD34+ selected,were cultured in the presence of Stem Cell Factor (SCF), GM-CSF, andTNF-α in endotoxin free RPMI 1640 medium (GIBCO, Grand Island, N.Y.)supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS;Flow Laboratories, Irvine, Calif.), 10 mM HEPES, 2 mM L-glutamine,5×10⁻⁵ M 2-mercaptoethanol, penicillin (100 μg/ml). This is referred toas complete medium.

[0177] CD34+ cells were seeded for expansion in 25 to 75 cm² flasks(Corning, N.Y.) at 2×10⁴ cells/ml. Optimal conditions were maintained bysplitting these cultures at day 5 and 10 with medium containing freshGM-CSF and TNF-α (cell concentration: 1-3×10⁵ cells/ml). In certaincases, cells were FACS sorted for CD1a expression at about day 6.

[0178] In certain situations, cells were routinely collected after 12days of culture, eventually adherent cells were recovered using a 5 μMEDTA solution. In other situations, the CD1a+ cells were activated byresuspension in complete medium at 5×10⁶ cells/ml and activated for theappropriate time (e.g., 1 or 6 h) with 1 μg/ml phorbol 12-myristate13-acetate (PMA, Sigma) and 100 ng/ml ionomycin (Calbiochem, La Jolla,Calif.). These cells were expanded for another 6 days, and RNA isolatedfor cDNA library preparation.

[0179] III. RNA Isolation and Library Construction

[0180] Total RNA is isolated using, e.g., the guanidine thiocyanate/CsClgradient procedure as described by Chirgwin, et al. (1978) Biochem.18:5294-5299.

[0181] Alternatively, poly(A)+ RNA is isolated using the OLIGOTEX mRNAisolation kit (QIAGEN). Double stranded cDNA are generated using, e.g.,the SUPERSCRIPT plasmid system (Gibco BRL, Gaithersburg, Md.) for cDNAsynthesis and plasmid cloning. The resulting double stranded cDNA isunidirectionally cloned, e.g., into pSport1 and transfected byelectroporation into ELECTROMAX DH10BTM Cells (Gibco BRL, Gaithersburg,Md.).

[0182] IV. Sequencing

[0183] DNA isolated from randomly picked clones, or after subtractivehybridization using unactivated cells, were subjected to nucleotidesequence analysis using standard techniques. A Taq DiDeoxy Terminatorcycle sequencing kit. (Applied Biosystems, Foster City, Calif.) can beused. The labeled DNA fragments are separated using a DNA sequencing gelof an appropriate automated sequencer. Alternatively, the isolated cloneis sequenced as described, e.g., in Maniatis, et al. (1982) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor Press; Sambrook, et al. (1989) Molecular Cloning: A LaboratoryManual, (2d ed.), vols. 1-3, CSH Press, NY; Ausubel, et al., Biology,Greene Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987and Supplements) Current Protocols in Molecular Biology, Greene/Wiley,New York. Chemical sequencing methods are also available, e.g., usingMaxam and Gilbert sequencing techniques.

[0184] V. Isolation of Human Monocyte Protein Genes

[0185] The FDF03, the YE01, and KTE03 (YYB01 and YYB04) clones weresequenced, and analyzed for open reading frames. The clones were furtheranalyzed to extend the nucleic acid sequence to a full, or nearly full,open reading frame.

[0186] mRNA is prepared from appropriate cell populations by theFastTrack kit (Invitrogen) from which cDNA is generated using, e.g.,SuperScript Plasmid System for cDNA synthesis from GIBCO-BRL(Gaithersburg, Md.) essentially as described by the manufacturer.Modification to the procedure may include the substitution of othercloning adapters for the Sal1 adapters provided with the kit. Theresultant cDNA from these cells is used to generate libraries, e.g., inthe plasmid PCDNA II (Invitrogen). The cDNA is cloned into thepolylinker and is used to transform an appropriate strain, e.g., DH10B,of E. coli. Plasmid is isolated and purified, e.g., with the Qiagensystem (Chatsworth, Calif.) which is used to generate RNA probes from,e.g., the SP6 promoter.

[0187] RNA probes are labeled, e.g., using the Genius System(Boehringer-Mannheim) as described by the manufacturer. Filter lifts ofthe cDNA library can be pre-hybridized, e.g., at 42° C. for 3-6 hours inChurch's buffer (50% formamide, 6×SSPE, 50 mM NaHPO₄ pH 7.2, 7% SDS,0.1% N-Lauryl sarcosine, 2% Boehringer-Mannheim blocking reagent).Filters are probed, e.g., overnight in the same buffer containing theappropriate probes. The filters are washed, e.g., as described by theGenius System. The colonies that hybridize are selected.

[0188] The entire cDNA of human monocyte proteins are sequenced, e.g.,by the dideoxynucleotide chain termination method with T7 polymerase(U.S. Biochemicals, Cleveland, Ohio) using double-stranded DNA astemplate. Data base searching and sequence analysis are performed usingIntelliGenetics programs (Mountain View, Calif.) to determine ifhomology exists between previously reported clones.

[0189] Table 1 discloses sequence encoding a human FDF03 gene and mousecounterpart sequence, and also shows alignment of available sequence.Likewise, Table 2 discloses three sequences encoding human YE01 geneproducts, including a splice variant and a transcript which encodes asoluble product. Table 3 provides sequences of embodiments of the KTE03gene products, and shows evidence of splice variants.

[0190] VI. Recombinant Monocyte Gene Constructs

[0191] Poly(A)⁺ RNA is isolated from appropriate cell populations, e.g.,using the FastTrack mRNA kit (Invitrogen, San Diego, Calif.). Samplesare electrophoresed, e.g., in a 1% agarose gel containing formaldehydeand transferred to a GeneScreen membrane (NEN Research Products, Boston,Mass.). Hybridization is performed, e.g., at 65° C. in 0.5 M NaHPO₄ pH7.2, 7% SDS, 1 mM EDTA, and 1% BSA (fraction V) with ³²P-dCTP labeledmonocyte gene cDNA at 10⁷ cpm/ml. After hybridization filters are washedthree times at 50° C. in 0.2×SSC, 0.1% SDS, and exposed to film for 24h.

[0192] The recombinant gene construct may be used to generate probe fordetecting the message. The insert may be excised and used in thedetection methods described above.

[0193] VII. Expression of Monocyte Gene Protein in E. coli.

[0194] PCR is used to make a construct comprising the open readingframe, preferably in operable association with proper promoter,selection, and regulatory sequences. The resulting expression plasmid istransformed into an appropriate, e.g., the Topp5, E. coli strain(Stratagene, La Jolla, Calif.). Ampicillin resistant (50 μg/ml)transformants are grown in Luria Broth (Gibco) at 37° C. until theoptical density at 550 nm is 0.7. Recombinant protein is induced with0.4 mm isopropyl-βD-thiogalacto-pyranoside (Sigma, St. Louis, Mo.) andincubation of the cells continued at 20° C. for a further 18 hours.Cells from a 1 liter culture are harvested by centrifugation andresuspended, e.g., in 200 ml of ice cold 30% sucrose, 50 mM Tris HCl pH8.0, 1 mM ethylenediamine-tetraacetic acid. After 10 min on ice, icecold water is added to a total volume of 2 liters. After 20 min on ice,cells are removed by centrifugation and the supernatant is clarified byfiltration via a 5 μM Millipak 60 (Millipore Corp., Bedford, Mass.).

[0195] The recombinant protein is purified via standard purificationmethods, e.g., various ion exchange chromatography methods.Immunoaffinity methods using antibodies described below can also beused. Affinity methods may be used where an epitope tag is engineeredinto an expression construct.

[0196] VIII. Mapping of Human Monocyte Genes

[0197] DNA isolation, restriction enzyme digestion, agarose gelelectrophoresis, Southern blot transfer and hybridization are performedaccording to standard techniques. See Jenkins, et al. (1982) J. Virol.43:26-36. Blots may be prepared with Hybond-N nylon membrane (Amersham).The probe is labeled with ³²P-dCTP; washing is done to a finalstringency, e.g., of 0.1×SSC, 0.1% SDS, 65° C.

[0198] Alternatively, a BIOS Laboratories (New Haven, Conn.) mousesomatic cell hybrid panel may be combined with PCR methods.

[0199] IX. Analysis of Individual Variation

[0200] From the distribution data, an abundant easily accessible celltype is selected for sampling from individuals. Using PCR techniques, alarge population of individuals are analyzed for this gene. cDNA orother PCR methods are used to sequence the corresponding gene in thedifferent individuals, and their sequences are compared. This indicatesboth the extent of divergence among racial or other populations, as wellas determining which residues are likely to be modifiable withoutdramatic effects on function.

[0201] X. Preparation of Antibodies

[0202] Recombinant monocyte proteins are generated by expression in E.coli as shown above, and tested for biological activity. Active ordenatured proteins may be used for immunization of appropriate mammalsfor either polyclonal serum production, or for monoclonal antibodyproduction. Antibodies are selected for use in Western blots, againstnative or denatured antigen, and for those which modulate a biologicalactivity.

[0203] Antibodies prepared against the FDF03 have confirmed specificbinding on dendritic cells. XI. Isolation of counterpart primatemonocyte genes Human cDNA clones encoding these genes are used asprobes, or to design PCR primers to find counterparts in various primatespecies, e.g., chimpanzees.

[0204] XII. Use of Reagents to Analyze Cell Populations

[0205] Detection of the level of monocyte cells present in a sample isimportant for diagnosis of certain aberrant disease conditions. Forexample, an increase in the number of monocytes in a tissue or the lymphsystem can be indicative of the presence of a monocyte hyperplasia,tissue or graft rejection, or inflammation. A low monocyte populationcan indicate an abnormal reaction to, e.g., a bacterial or viralinfection, which may require the appropriate treat to normalize themonocyte response.

[0206] FACS analysis using a labeled binding agent specific for a cellsurface monocyte protein, see, e.g., Melamed, et al. (1990) FlowCytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988)Practical Flow Cytometry Liss, New York, N.Y.; and Robinson, et al.(1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y., isused in determining the number of monocytes present in a cell mixture,e.g., PBMCs, adherent cells, etc. The binding agent is also used forhistological analysis of tissue samples, either fresh or fixed, toanalyze infiltration of monocyte. Diverse cell populations may also beevaluated, either in a cell destructive assay, or in certain assayswhere cells retain viability.

[0207] Analysis of the presence of soluble intracellular molecules isperformed, e.g., with a fluorescent binding agent specific for amonocyte as described in Openshaw, et al. (1995) J. Exp. Med.182:1357-1367. alternatively, tissue or cell fixation methods may beused.

[0208] Levels of monocyte transcripts are quantitated, e.g., usingsemiquantitative PCR as described in Murphy, et al. (1993) J. Immunol.Methods 162:211-223. Primers are designed such that genomic DNA is notdetected.

[0209] Distribution of the FDF03 embodiment has been studied usinghybridization and PCR analysis. Northern blot analysis locatedtranscripts in dendritic cells and the JY cell line. There appear to betwo transcripts of about 700 bp and 1300 bp, which may be differentiallyregulated, and an estimated frequency of about 1 in 4000 in restingmonocytes or LPS and IFNγ activated monocytes. The shorter message doesnot appear to encode a soluble version of the protein, e.g., lacking theTM and intracellular segments. Southern blot analysis has detectedtranscripts in monocytes, dendritic cells, PBMC, B cells, and splenic Bcells. The message appears to be down-regulated upon monocyteactivation.

[0210] Distribution of the YE01 embodiment has also been evaluated. Themessage appears to be monocyte specific, and is a low abundance message.It is detectable in cDNA Southern blots in resting monocytes, and inactivated monocytes. Its highest expression was found in 6 hour LPSstimulated monocytes. It is also detectable in anti-CD3 and PMAactivated PBMC. It may be faintly detectable in dendritic cells, butthis may be due to contamination of the dendritic cell population withresidual monocytes. At that level of sensitivity, it is undetectable inNK cells, B or T cells, or any fetal cells examined. However, the YE01gene product is specifically recognized by a monoclonal antibody DX26.This antibody, when crosslinked, can inhibit NK cell mediated killing ofcertain targets. The antibody recognizes protein expressed in T cells, Bcells, NK cells, and monocytes. The gene encoding the antigen recognizedby DX26, which is apparently a polymorphic variant of the YE01 isolate,has been cloned and has essentially the sequence:

[0211] The KTE03 expression levels were also investigated. The messageappeared to be up-regulated upon IL-10 exposure when the monocytes wereactivated by LPS and IFNγ.

[0212] XIII. Isolation of a Binding Counterpart

[0213] A monocyte protein can be used as a specific binding reagent, bytaking advantage of its specificity of binding, much like an antibodywould be used. A binding reagent is either labeled as described above,e.g., fluorescence or otherwise, or immobilized to a substrate forpanning methods.

[0214] The monocyte protein is used to screen for a cell line whichexhibits binding. Standard staining techniques are used to detect orsort intracellular or surface expressed ligand, or surface expressingtransformed cells are screened by panning. Screening of intracellularexpression is performed by various staining or immunofluorescenceprocedures. See also McMahan, et al. (1991) EMBO J. 10:2821-2832.

[0215] For example, on day 0, precoat 2-chamber permanox slides with 1ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min at roomtemperature. Rinse once with PBS. Then plate COS cells at 2-3×10⁵ cellsper chamber in 1.5 ml of growth media. Incubate overnight at 37° C.

[0216] On day 1 for each sample, prepare 0.5 ml of a solution of 66mg/ml DEAE-dextran, 66 mM chloroquine, and 4 mg DNA in serum free DME.For each set, a positive control is prepared, e.g., of humanreceptor-FLAG cDNA at 1 and 1/200 dilution, and a negative mock. Rinsecells with serum free DME. Add the DNA solution and incubate 5 hr at 37°C. Remove the medium and add 0.5 ml 10% DMSO in DME for 2.5 min. Removeand wash once with DME. Add 1.5 ml growth medium and incubate overnight.

[0217] On day 2, change the medium. On days 3 or 4, the cells are fixedand stained. Rinse the cells twice with Hank's Buffered Saline Solution(HBSS) and fix in 4% paraformaldehyde (PFA)/glucose for 5 min. Wash 3×with HBSS. The slides may be stored at −80° C. after all liquid isremoved. For each chamber, 0.5 ml incubations are performed as follows.Add HBSS/saponin(0.1%) with 32 ml/ml of 1M NaN₃ for 20 min. Cells arethen washed with HBSS/saponin 1×. Add protein or protein/antibodycomplex to cells and incubate for 30 min. Wash cells twice withHBSS/saponin. If appropriate, add first antibody for 30 min. Add secondantibody, e.g., Vector anti-mouse antibody, at 1/200 dilution, andincubate for 30 min. Prepare ELISA solution, e.g., Vector Elite ABChorseradish peroxidase solution, and preincubate for 30 min. Use, e.g.,1 drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 mlHBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRP solutionand incubate for 30 min. Wash cells twice with HBSS, second wash for −2min, which closes cells. Then add Vector diaminobenzoic acid (DAB) for 5to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2 drops of H₂O₂per 5 ml of glass distilled water. Carefully remove chamber and rinseslide in water. Air dry for a few minutes, then add 1 drop of CrystalMount and a cover slip. Bake for 5 min at 85-90° C.

[0218] Alternatively, other monocyte protein specific binding reagentsare used to affinity purify or sort out cells expressing a receptor.See, e.g., Sambrook, et al. or Ausubel, et al.

[0219] Another strategy is to screen for a membrane bound receptor bypanning. The receptor cDNA is constructed as described above. The ligandcan be immobilized and used to immobilize expressing cells.Immobilization may be achieved by use of appropriate antibodies whichrecognize, e.g., a FLAG sequence of a monocyte protein fusion construct,or by use of antibodies raised against the first antibodies. Recursivecycles of selection and amplification lead to enrichment of appropriateclones and eventual isolation of ligand expressing clones.

[0220] Phage expression libraries can be screened by monocyte protein.Appropriate label techniques, e.g., anti-FLAG antibodies, will allowspecific labeling of appropriate clones.

[0221] XIV. Isolation of a Soluble YE01

[0222] An additional family member of the previously described YE01,also designated DNAX Leukocyte Associated Immunoglobulin-like Receptor(DLAIR; and now designated DLAIR-1) was cloned by screening a human Tcell tumor line cDNA library (TcT). Bacterial colony lift membranes werehybridized with a DLAIR-1 probe comprising a BglII-SphI digestionfragment, spanning the Ig loop in the extracellular domain. Twopositives were isolated and sequenced. Sequence analysis revealed thatboth clones contained identical open reading frames of 414 base pairs,encoding a 135 amino acid protein with a predicted 21 amino acid leadersequence and a predicted molecular weight of 14.7 kDa. This molecule,now referred to as DLAIR-2, contains one Ig loop. See Table 2. The Igloop has 84% homology with DLAIR-1, indicating that it belongs to thesame family, but is encoded by a separate gene. DLAIR-2 lacks atransmembrane region which suggests that it is a secreted protein.

[0223] DLAIR-2, as a soluble molecule with similarity to DLAIR-1, may beused as an antagonist to this inhibitory receptor.

[0224] XV. Preparation of DX26 Monoclonal Antibody

[0225] Mice were immunized with a human NK cell clone and antibodieswere screened for their capacity to inhibit NK cell-mediated lysis ofFcR bearing targets. Alternatively, antibodies will be raised topurified protein.

[0226] XVI. Cross-Linking DLAIR-1 with mAb Inhibits NK cell-mediatedKilling

[0227] DX26 mAb did not inhibit NK clone killing of the HLA-negativeEBV-transformed B cell line 721.221. However, when 721.221 wastransfected with the human FcγR-II (CD32) and used as a target, NKcell-mediated cytolysis was inhibited by DX26 mAb. This indicates thatsignaling through the molecule recognized by DX26 mAb (designated DNAXLeukocyte Associated Immunoglobulin-like Receptor (DLAIR)), delivers anegative signal to NK cell clones that prevents their killing specifictarget cells. In agreement with this, NK cell-mediated cytotoxicityagainst Colo-205, PA-1, or FO-1, each an FcR-negative human cell line,was not inhibited by the addition of DX26 mAb. Moreover, lysis of P815cells, an FcR-expressing mouse mastocytoma cell line, which is killed invitro by human NK cell clones upon simultaneous cross linking of CD2,CD16, CD69, or DNAM-1 antigen, was also inhibited by DX26 mAb. Theseresults lead to a conclusion that DLAIR delivers a strong inhibitorysignal to NK cells, since the positive signal given by potent inducersof NK cell cytotoxicity was overruled by DX26 mAb.

[0228] XVII. DLAIR-1 is an Inhibitory Receptor on Resting NK Cells

[0229] NK cell clones consist of clonally derived populations ofactivated NK cells. These cells are potently inhibited by DLAIRsignaling. We set out to study whether DLAIR is also functioning as aninhibitory receptor on NK cells that had not been previously activated.Resting NK cells, prepared from peripheral blood by negative depletionusing magnetic beads, were able to lyse P815 target cells whensimultaneously activated through CD16. This NK cell mediatedcytotoxicity was inhibited by the addition of DX26 mAb. Thus, DLAIR isfunctional as an inhibitory receptor on both activated and resting NKcells.

[0230] XVIII. DLAIR is a Widely Expressed Antigen

[0231] Phenotypic analysis of human peripheral blood lymphocytesdemonstrated that DLAIR is a widely distributed molecule. In healthydonor PBMC, CD3⁺CD4⁺ T cells (70-80%), CD3⁺CD8⁺T cells (80-90%),CD3⁻CD56⁺ NK cells (95-100%), CD3⁻CD19⁺ B cells (80-90%), and CD3⁻CD14+monocytes (99-100%) all expressed the DLAIR molecule. Human fetalthymocytes, both the immature CD4+CD8+ cells and mature CD4+-CD8⁺ orCD4⁻CD8⁺ single positive cells also expressed DLAIR. Peripheral bloodgranulocytes, platelets and erythrocytes did not express DLAIR.

[0232] Human NK cell clones and T cell clones all expressed DLAIR, withthe exception of the long-term cultured NK clones NKL and NK92 (seeTable 4). EBV-transformed B cell lines, the B cell tumor Daudi, and theNK tumor cell line YT and several non-hematopoietic cell lines did notexpress DLAIR, whereas human T cell lines did show DLAIR expression.TABLE 4 Expression of DLAIR on human tumor cell lines¹ control IgG1 DX26mAb cell line type (mean fluorescence intensity) HUT78 T cell tumor <525.8 Peer T cell tumor <5 29.1 Molt4 T cell tumor <5 30.7 CEM T celltumor <5 92.7 Jurkat T cell tumor <5 47.1 HL60 promyeloid tumor <5 46.9U937 myeloid tumor <5 49.5 721.221 EBV - B cell <5 <5 JY EBV - B cell <5<5 Daudi B cell tumor <5 <5 YT NK cell tumor <5 <5 NKL NK cell clone <5<5 NK92 NK cell clone <5 <5 Colo205 colon carcinoma <5 <5 293T embryonickidney <5 <5 PA-1 teratocarcinoma <5 <5 FO-1 melanoma <5 <5

[0233] XIX. Expression Cloning of the DX26 Antigen

[0234] The DX26 antibody was used to expression clone the antigen theantibody recognizes. The expression cloning was performed using standardmethods. See, e.g., Sambrook, et al. or Coligan, et al.

[0235] DX26 antigen is expression cloned, e.g., from a polyclonal humanactivated NK cell cDNA library in the pJFE14 expression vector. COS7cells are transfected with the library and antigen positive cells wereselected using phycoerythrin labeled anti-DX26 mAb. The cDNA sequencewas determined and found to match much of the YE01 sequence. The DX26antibody specifically binds to the product of the YE01 gene product.

[0236] In another method, oligonucleotides are used to screen a library.In combination with polymerase chain reaction (PCR) techniques,synthetic oligonucleotides in appropriate orientations are used asprimers to select correct clones from a library.

[0237] Moreover, the YE01 gene product is specifically recognized by amonoclonal antibody DX26. This antibody, when crosslinked, can inhibitNK cell mediated killing of certain targets. The antibody recognizesprotein expressed in T cells, B cells, NK cells, and monocytes. The geneencoding the antigen recognized by DX26, which is apparently apolymorphic variant of the YE01 isolate, has been cloned and hasessentially the sequence (see SEQ ID NO: 7). This isolate has adifferent 3′ untranslated sequence from the original YE01 transcript,apparently due to use of an alternative polyadenylation site. A solubleform of DLAIR has also been detected (see SEQ ID NO: 9).

[0238] Distribution analysis of the DX26 isolate has determined,Northern blot analysis, the distribution as follows. Probing of mRNA ofhuman NK cell clones with DLAIR cDNA, PBMC, the human T cell lineJurkat, and the human myeloid cell line Jurkat results in two bands ofapproximately 1800 bp and 3000-4000 bp. This indicates that besides thecloned cDNA, another transcript with sequence similarity to DLAIR existsin these cell lines. Whether this contains the same open reading frameis at present unknown, but will be determined upon cloning and sequenceanalysis of that transcript. The EBV-transformed human B cell line JYdid not show transcripts that probed with DLAIR cDNA.

[0239] XX. DLAIR-1 Binds SHP-1 and SHP-2

[0240] The existence of two consensus sequences for ITIMs within thecytoplasmic domain of DLAIR-1, suggested that the generation of aninhibitory signal in NK cells was manifested by the recruitment of SHP-1and/or SHP-2. To determine if DLAIR-1 was capable of binding proteintyrosine phosphatases, a NK cell clone was stimulated with pervanadate(an inhibitor of protein tyrosine phosphatases that induces tyrosinephosphorylation (O'Shea, et al. (1992) Proc. Natl. Acad. Sci. USA89:10306-10310), lysed, and immunoprecipitated with DX26 MAb.Immunoprecipitates were then analyzed by Western blot using antibodiesspecific for SHP-1 and SHP-2. Both SHP-1 and SHP-2 associated withtyrosine phosphorylated DLAIR-1. These results suggest that recruitmentof SHP-1 and SHP-2 may be involved in mediating the negative signaltransduced via engagement of the DLAIR-1 molecule.

[0241] All references cited herein are incorporated herein by referenceto the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety for all purposes.

[0242] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

What is claimed is:
 1. A composition of matter selected from the groupconsisting of: a) a substantially pure or recombinant FDF03 protein orpeptide exhibiting at least about 85% sequence identity over a length ofat least about 12 amino acids to a mature polypeptide from SEQ ID NO: 2or 4; b) a natural sequence FDF03 of SEQ ID NO: 2 or 4; c) a fusionprotein comprising FDF03 sequence; d) a substantially pure orrecombinant YE01 protein or peptide exhibiting at least about 85%sequence identity over a length of at least about 12 amino acids to amature polypeptide from SEQ ID NO: 6, 8, or 10; e) a natural sequenceYE01 of SEQ ID NO: 6, 8, or 10; f) a fusion protein comprising YE01sequence; g) a substantially pure or recombinant KTE03 protein orpeptide exhibiting at least about 85% sequence identity over a length ofat least about 12 amino acids to SEQ ID NO: 12, 14, 16, 18, 20, or 22;h) a natural sequence KTE03 of SEQ ID NO: 12, 14, 16, 18, 20, or 22; andi) a fusion protein comprising KTE03 sequence.
 2. A substantially pureor isolated protein comprising a segment exhibiting sequence identity toa corresponding portion of a FDF03, YE01, or KTE03 of claim 1, wherein:a) said homology is at least about 90% identity and said portion is atleast about 9 amino acids; b) said homology is at least about 80%identity and said portion is at least about 17 amino acids; or c) saidhomology is at least about 70% identity and said portion is at leastabout 25 amino acids.
 3. The composition of matter of claim 1, whereinsaid: a) FDF03 comprises a mature sequence of Table 1; b) YE01 comprisesa mature sequence of Table 2; c) KTE03 comprises a mature sequence ofTable 3; d) protein or peptide: i) is from a warm blooded animalselected from a mammal, including a primate or rodent; ii) comprises atleast one polypeptide segment of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, or 22; iii) exhibits a plurality of portions exhibiting saididentity; iv) is a natural allelic variant of FDF03, YE01, or KTE03; v)has a length at least about 30 amino acids; vi) exhibits at least twonon-overlapping epitopes which are specific for a mammalian FDF03, YE01,or KTE03; vii) exhibits a sequence identity at least about 90% over alength of at least about 20 amino acids to a rodent FDF03, YE01, orKTE03; viii) exhibits at least two non-overlapping epitopes which arespecific for a primate FDF03, YE01, or KTE03; ix) exhibits a sequenceidentity at least about 90% over a length of at least about 20 aminoacids to a primate FDF03, YE01, or KTE03; x) is glycosylated; xi) has amolecular weight of at least 7 kD with natural glycosylation; xii) is asynthetic polypeptide; xiii) is attached to a solid substrate; xiv) isconjugated to another chemical moiety; xv) is a 5-fold or lesssubstitution from natural sequence; or xvi) is a deletion or insertionvariant from a natural sequence.
 4. A composition comprising: a) asterile FDF03 protein or peptide of claim 1; b) said FDF03 protein orpeptide of claim 1 and a carrier, wherein said carrier is: i) an aqueouscompound, including water, saline, and/or buffer; and/or ii) formulatedfor oral, rectal, nasal, topical, or parenteral administration; c) asterile YE01 protein or peptide of claim 1; d) said YE01 protein orpeptide of claim 1 and a carrier, wherein said carrier is: i) an aqueouscompound, including water, saline, and/or buffer; and/or ii) formulatedfor oral, rectal, nasal, topical, or parenteral administration; e) asterile KTE03 protein or peptide of claim 1; or f) said KTE03 protein orpeptide of claim 1 and a carrier, wherein said carrier is: i) an aqueouscompound, including water, saline, and/or buffer; and/or ii) formulatedfor oral, rectal, nasal, topical, or parenteral administration.
 5. Thefusion protein of claim 1, comprising: a) mature protein sequence ofTable 1, 2, or 3; b) a detection or purification tag, including a FLAG,His6, or Ig sequence; or c) sequence of another cell surface protein. 6.A kit comprising a protein or polypeptide of claim 1, and: a) acompartment comprising said protein or polypeptide; and/or b)instructions for use or disposal of reagents in said kit.
 7. A bindingcompound comprising an antigen binding portion from an antibody, whichspecifically binds to a natural FDF03, YE01, or KTE03 protein of claim1, wherein: a) said protein is a rodent protein; b) said bindingcompound is an Fv, Fab, or Fab2 fragment; c) said binding compound isconjugated to another chemical moiety; or d) said antibody: i) is raisedagainst a peptide sequence of a mature polypeptide of Table 1, 2, or 3;ii) is raised against a mature FDF03, YE01, or KTE03; iii) is raised toa purified FDF03, YE01, or KTE03; iv) is immunoselected; v) is apolyclonal antibody; vi) binds to a denatured FDF03, YE01, or KTE03;vii) exhibits a Kd to antigen of at least 30 μM; viii) is attached to asolid substrate, including a bead or plastic membrane; ix) is in asterile composition; or x) is detectably labeled, including aradioactive or fluorescent label.
 8. A kit comprising said bindingcompound of claim 7, and: a) a compartment comprising said bindingcompound; and/or b) instructions for use or disposal of reagents in saidkit.
 9. The kit of claim 8 capable of making a qualitative orquantitative analysis.
 10. A composition comprising: a) a sterilebinding compound of claim 7; or b) said binding compound of claim 7 anda carrier, wherein said carrier is: i) an aqueous compound, includingwater, saline, and/or buffer; and/or ii) formulated for oral, rectal,nasal, topical, or parenteral administration.
 11. An isolated orrecombinant nucleic acid encoding a protein or peptide or fusion proteinof claim 1, wherein: a) said protein is from a mammal, including aprimate; or b) said nucleic acid: i) encodes an antigenic peptidesequence of Table 1, 2, or 3; ii) encodes a plurality of antigenicpeptide sequences of Table 1, 2, or 3; iii) exhibits at least about 80%identity to a natural cDNA encoding said segment; iv) is an expressionvector; v) further comprises an origin of replication; vi) is from anatural source; vii) comprises a detectable label; viii) comprisessynthetic nucleotide sequence; ix) is less than 6 kb, preferably lessthan 3 kb; x) is from a mammal, including a primate; xi) comprises anatural full length coding sequence; xii) is a hybridization probe for agene encoding said protein; or xiii) is a PCR primer, PCR product, ormutagenesis primer.
 12. A cell or tissue comprising a recombinantnucleic acid of claim
 11. 13. The cell of claim 12, wherein said cellis: a) a prokaryotic cell; b) a eukaryotic cell; c) a bacterial cell; d)a yeast cell; e) an insect cell; f) a mammalian cell; g) a mouse cell;h) a primate cell; or i) a human cell.
 14. A kit comprising said nucleicacid of claim 11, and: a) a compartment comprising said nucleic acid; b)a compartment further comprising a FDF03, YE01, or KTE03 protein orpolypeptide; and/or b) instructions for use or disposal of reagents insaid kit.
 15. The kit of claim 14 capable of making a qualitative orquantitative analysis.
 16. A nucleic acid which: a) hybridizes underwash conditions of 30° C. and less than 2M salt to the coding portionfrom SEQ ID NO: 1 or 3; b) hybridizes under wash conditions of 30° C.and less than 2 M salt to the coding portion from SEQ ID NO: 5, 7, or 9;c) hybridizes under wash conditions of 30° C. and less than 2M salt tothe coding portion from SEQ ID NO: 11, 13, 15, 17, 19, or 21; d)exhibits at least about 85% identity over a stretch of at least about 30nucleotides to a primate FDF03; e) exhibits at least about 85% identityover a stretch of at least about 30 nucleotides to a primate YE01; or f)exhibits at least about 85% identity over a stretch of at least about 30nucleotides to a primate KTE03.
 17. The nucleic acid of claim 16,wherein: a) said wash conditions are at 45° C. and/or 500 mM salt; or b)said identity is at least 90% and/or said stretch is at least 55nucleotides.
 18. The nucleic acid of claim 17, wherein: a) said washconditions are at 55° C. and/or 150 mM salt; or b) said identity is atleast 95% and/or said stretch is at least 75 nucleotides.
 19. A methodof modulating physiology or development of a cell or tissue culture cellcomprising contacting said cell with an agonist or antagonist of aFDF03, YE01, or KTE03.
 20. The method of claim 19, wherein the cell is aleukocyte, and the antagonist is to YE01 and is a monoclonal antibodywhich binds to DLAIR-1.